Class of HDAC Inhibitors Expands the Renal Progenitor Cells Population and Improves the Rate of Recovery from Acute Kidney Injury

ABSTRACT

Compounds and compositions are provided that inhibit histone deacylase activity and which expand renal progenitor cell populations and improve kidney function in a damaged kidney. Methods of use of the compounds and compositions are provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.13/982,512, filed Nov. 12, 2013, which is a national stage applicationof International Patent Application No. PCT/US2012/024625, filed Feb.10, 2012, which claims the benefit of U.S. Provisional Application No.61/441,560, filed Feb. 10, 2011, entitled “Class of HDAC InhibitorsExpands the Renal Progenitor Cells Population and Improves the Rate ofRecovery from Acute Kidney Injury,” each of which is incorporated hereinby reference in its entirety.

STATEMENT REGARDING FEDERAL FUNDING

This invention was made with government support under Grant Nos.DK069403 and DK053287 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

The Sequence Listing associated with this application is filed inelectronic format via EFS-Web and is hereby incorporated by referenceinto the specification in its entirety. The name of the text filecontaining the Sequence Listing is 6527_1801643_ST25.txt. The size ofthe text file is 2,427 bytes, and the text file was created on Apr. 23,2018.

Severe acute kidney injury (AKI) is remarkably common and has anunacceptably high mortality that has been unchanged for the last twentyyears. AKI therapies that have been developed in experimental modelswhen administered prior to the onset of injury have failed to showtherapeutic benefit in humans. However, the kidney has an innatecapacity to undergo epithelial regeneration following injury, suggestingthat drugs that enhance this regenerative capacity are more likely to beof benefit when given after the onset of injury.

AKI is a multi-factorial disorder that occurs in approximately 7% ofin-patients hospital admissions. It is an independent predictor ofin-hospital mortality. Severe AKI requiring renal replacement therapyoccurs in 4% of critically ill patients and has 50% in-patientmortality. Long term follow up studies in survivors of severe AKIindicate that approximately 12.5% become dialysis-dependent. Despitethese sobering statistics, renal replacement is the only approvedtherapy for AKI, and there are no established therapies that have beenproven to prevent renal injury or accelerate the rate of renal recoveryfollowing induction of AKI in man Therefore there is an urgent need todevelop effective therapies that will accelerate the rate of recoveryfollowing induction of renal injury.

SUMMARY

The studies below identify novel, non-toxic therapeutic agents thataccelerate recovery from AKI by enhancing the innate regenerativecapacity of the kidney, thereby identifying a number of lead compoundswith a high probability of targeted translatability for application inhuman AKI. A state of the art, high content functional screen usingtransgenic zebrafish embryos was developed to identify compounds thatcause expansion of embryonic renal progenitor cells. This strategy wasvalidated by identifying a unique and novel class of histone deacetylaseinhibitors (HDACi) that increase the rate of recovery in a mouse modelof AKI. These findings point to a promising role for this class of smallmolecules in patients with AKI.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. HDACi Analogs. Lead compound, PTBA is shown in the center.Potential HDACi analogue families include: left arrow, esterifiedcompounds; upper left arrow, esterified compounds with bulk added to thephenol ring for increased Log P; upper right arrow, hydroxamic compoundswith bi-dentate zinc chelator; right arrow, hydroxamic compounds withbulk added to the phenol ring for increased Log P; downward arrow, otherproven HDACi, such as SAHA and TSA.

FIG. 2. Four compounds causing pericardial edema in the initialphenotypic screen. (A through D) Top panels: chemical structures ofNSC115787 (A), NSC134664 (B), NSC357777 (C), or NSC35400 (D). Bottompanels: edemic phenotypes in 72 hours post-fertilization (hpf) larvaetreated with the corresponding compounds at 10 μM.

FIG. 3. PTBA elicits concentration-dependent effects on larval edema andsurvival. (A through D) Embryos were treated with 0 to 10 μM PTBA from 2hpf, and larvae were scored at 72 hpf using a phenotype-basedclassification system (see Methods). (A) Wild-type (WT). (B) Edemic 1(E1). (C) Edemic 2 (E2). (D) Edemic 3 (E3). (E) Graph of phenotypesafter treatment with 0 to 10 μM PTBA (n=90 per concentration). Asteriskdenotes lowest PTBA concentration that exhibits a significant effect(p<0.05) on survival.

FIG. 4. PTBA treatment increases the expression of renal progenitormarkers. (A through F) In situ hybridization for lhx1a (A and B), pax2a(C and D), or pax8 (E and F), in 10-somite embryos treated with 0.5%DMSO (A, C, and E) or 3 μM PTBA (B, D, and F). Arrowheads indicate renalprogenitor cells, asterisk indicates notochord. Relative qPCR in thetrunk region of 10-somite embryos (n=4, 60 embryos per group) isdisplayed under the corresponding in situ image. Data are meanexpression with 95% confidence interval, determined as described below.

FIG. 5. PTBA treatment increases the number of renal progenitor cells.(A through H) Confocal projections of 10-somite Tg(lhx1a:EGFP)pt303embryos treated with 0.5% DMSO (n=18 [A through D] or 3 μM PTBA (n=21 [Ethrough H]). Boxed areas (A and E) were counted for GFP- andDAPI-positive nuclei and are shown in B and F (GFP), C and G (DAPI), andD and H (merge). Cell counts are mean number of positive cells plus 95%confidence interval (A and E).

FIG. 6. PTBA is effective during renal progenitor cell specification. (Athrough G) In situ hybridization for pax2a in 24 hpf embryos treatedwith 0.5% DMSO from 2 hpf (A, n=132) or 3 μM PTBA from: 2 hpf (B, n=67),shield (C, 6 hpf, n=100), 2 somites (D, 10.7 hpf, n=71), 5 somites (E,11.7 hpf, n=87), 10 somites (F, 14 hpf, n=89), or 15 somites (G, 16.5hpf, n=72). Insets are pax2a enlargements (lower field).

FIG. 7. PTBA treatment expands cdh17 expression. (A through D) In situhybridization for cdh17 expression in 48 hpf embryos treated with 0.5%DMSO (A [magnified in B]) or 3 μM PTBA (C [magnified in D]). (E and F)Proximal tubule cross-sections (5 μm) taken from cdh17 in situhybridizations of 48 hpf embryos treated with 0.5% DMSO (E) or 3 μM PTBA(F). Cross-sections were taken from the locations indicated in B and Dby yellow lines.

FIG. 8. PTBA treatment expands NaK-ATPase expression. (A and B)Whole-mount antibody staining for NaK-ATPase in 48 hpf embryos treatedwith 0.5% DMSO (A) or 3 μM PTBA (B). (C and D) Distal tubulecross-sections (5 μm) taken from NaK-ATPase antibody stained 48 hpfembryos treated with 0.5% DMSO (C) or 3 μM PTBA (D). White arrowheadsindicate NaK-ATPase protein expression. Cross-sections were taken fromthe locations indicated in A and B by yellow lines.

FIG. 9. PTBA treatment expands several pronephric regions. (A through F)In situ hybridization for the podocyte marker wt1a (A and B), theproximal tubule marker slc4a4 (C and D) and the distal tubule markerslc12a1 (E and F) in 48 hpf embryos treated with 0.5% DMSO (A, C, and E)or 3 μM PTBA (B, D, and F). Brackets in A and B indicate the expressiondomain of wt1a.

FIG. 10. PTBA treatment does not transform nearby tissues to a renalfate. (A through F) In situ hybridization for the mesodermal markersmyod1 (A and B), fli1a (C and D), and ntla (E and F) in 10-somiteembryos treated with 0.5% DMSO (A, C, and E) or 3 μM PTBA (B, D, and F).Relative mRNA abundance in the trunk region of 10-somite embryos (n=4,60 embryos per group) is displayed under the corresponding in situimage. Data are mean expression with 95% confidence interval, determinedas described below.

FIG. 11. PTBA requires proliferation for efficacy. (A through D) In situhybridization for lhx1a expression in 10-somite embryos treated at 5 hpfwith 0.5% DMSO (A and B) or HUA (C and D). At 8 hpf, treatment solutionswere replaced with 0.5% DMSO (A), 3 μM PTBA (B), HUA (C), or 3 μM PTBAand HUA (D).

FIG. 12. Synthesis scheme for PTBA

FIG. 13. Structure-activity relationship studies reveal essentialmoieties for PTBA efficacy. (A through I) In situ hybridization forlhx1a expression in 10-somite embryos treated with 0.5% DMSO (A) or 3 μMof the following compounds: PTBA (B), 4-(phenylsulfonyl)butanoic acid(PSOBA) (C), 4-(naphthalen-2-ylthio)butanoic acid (D), 2-amino-PTBA (E),3-(phenylthio)benzoic acid (F), 4-phenoxybutanoic acid (G),5-phenylpentanoic acid (H), and methyl-4-(phenylthio)butanoate (I).

FIG. 14. PTBA exhibits structural similarity to HDACis. (A through D)Structures of PTBA (A), PBA (B), TSA (C), and the general HDACipharmacophore (D) containing a cap (CAP), connecting unit (U),hydrophobic linker (LINKER), and zinc-binding group (ZBG).

FIG. 15. Treatment with the HDACi PBA expands the kidney field. (Athrough F) In situ hybridization for lhx1a in 10-somite embryos treatedfrom 2 hpf with: 0.5% DMSO (A, n=59), 10 μM PBA (B, n=58), 15 μM PBA (C,n=60), 20 μM PBA (D, n=59), 25 μM PBA (E, n=58), or 30 μM PBA (F, n=53).

FIG. 16. Treatment with the HDACi TSA expands the kidney field. (Athrough F) In situ hybridization for lhx1a in 10-somite embryos treatedfrom 2 hpf with: 0.5% DMSO (A, n=60), 100 nM TSA (B, n=57), 150 nM TSA(C, n=57), 200 nM TSA (D, n=52), 250 nM TSA (E, n=42), or 300 nM TSA (F,n=40).

FIG. 17. Treatment with PBA or TSA affects nearby tissues. (A through I)In situ hybridization for myod1 (A through C), fli1a (D through F), orntla (G through I) in 10-somite embryos treated with 0.5% DMSO (A, D,and G), 25 μM PBA (B, E, and H), or 200 nM TSA (C, F, and I). Arrowheadsindicate breaks in ntla expression.

FIG. 18. PBA elicits concentration-dependent effects on larval edema andsurvival. (A through D) Embryos were treated with 0 to 30 μM PBA from 2hpf, and larvae were scored at 72 hpf using a phenotype-basedclassification system described herein. (A) Wildtype (WT). (B) Edemic 1(E1). (C) Edemic 2 (E2). (D) Graph of phenotypes after treatment with 0to 30 μM PBA (n=90 per concentration). Asterisk denotes lowest PBAconcentration that exhibits a significant effect (p<0.05) on survival.

FIG. 19. TSA elicits concentration-dependent effects on larval edema andsurvival. (A through D) Embryos were treated with 0 to 300 nM TSA from 2hpf, and larvae were scored at 72 hpf using a phenotype-basedclassification system described herein. (A) Wildtype (WT). (B) Edemic 1(E1). (C) Edemic 2 (E2). (D) Edemic 3 (E3). (E) Graph of phenotypesafter treatment with 0 to 300 nM PBA (n=90 per concentration). Asteriskdenotes lowest TSA concentration that exhibits a significant effect(p<0.05) on survival.

FIG. 20. PTBA functions as an HDACi in vitro. Fluorescence histonedeacetylation assay performed in the presence of 5 mM PTBA, 5 mM PBA, 5mM PSOBA, 1 μM TSA, or 5% DMSO. At a given amount of nuclear extract,less fluorescence indicates less HDAC activity. Error bars represent the95% confidence intervals for each data point.

FIG. 21. PTBA functions as an HDACi in vivo. Western blot examining theacetylation state of histone H4 isolated from embryos at 30 hpf that hadbeen treated for 6 hours with 0.5% DMSO, 3 μM (1×) or 15 μM (5×) PTBA,25 μM (1×) or 125 μM (5×) PBA, and 200 nM (1×) or 1 μM (5×) TSA. Westernblot for α-tubulin demonstrates equal loading.

FIG. 22. PTBA affects the expression of RA-responsive genes. (A throughD) In situ hybridization for cyp26a1 (A and B) and cmlc2 (C and D) in18-somite embryos treated with 0.5% DMSO (A and C) or 3 μM PTBA (B andD). Arrowheads highlight cyp26a1 expression domains.

FIG. 23. RA signaling mediates PTBA efficacy. (A through D) In situhybridization in 10-somite embryos mock-injected with 1% fluoresceindextran (A and B) or injected with 200 pg of DN-RARα mRNA and 1%fluorescein dextran (C and D). At 5 hpf, embryos were treated with 0.5%DMSO (A and C) or 3 μM PTBA (B and D).

FIG. 24 General structure of a PTBA analog containing functional groupsubstitutions as described in Table 2.

FIG. 25. Functional analogs of PTBA. (A through J) Structures of theHDACis butanoic acid (A), valproic acid (B), 4-phenylbutanoic acid (PBA,C), trichostatin A (TSA, D), SAHA (E), APHA compound 8 (F), Scriptaid(G), MS-275 (H), apicidin (I), and tubacin (J).

FIG. 26. PTBA analogs exhibiting partial efficacy at 3 μM. (A through F)In situ hybridization for lhx1a expression in 10-somite embryos treatedfrom 2 hpf with 0.5% DMSO (A) or 3 μM of the following compounds:valproic acid (20% expansion) (B), Scriptaid (25% expansion) (C),N-hydroxy-4-[(4-methoxyphenyl)thio]butanamide (31% expansion) (D),tert-butyl 4-(phenylthio)butanoate (39% expansion) (E), and methyl4-[(bromophenyl)thio]butanoate (74% expansion) (F).

FIGS. 27A-27B. PTBA analogs exhibiting efficacy at 1.5 μM. (panels Athrough N) In situ hybridization for lhx1a expression in 10-somiteembryos treated from 2 hpf with 0.5% DMSO (A) or 1.5 μM of the followingcompounds: 4-[(bromophenyl)thio]-N-hydroxybutanamide (47% expansion)(B), 4-[(4-chlorophenyl)thio]butanoate (72% expansion) (C), PTBA (83%expansion) (D), N-hydroxy-4-(phenylthio)butanamide (92% expansion) (E),N-hydroxy-4-[(4-methylphenyl)thio]butanamide (97% expansion) (F),4[(4-chlorophenyl)thio]-Nhydroxybutanamide (97% expansion) (G),4-[(4-fluorophenyl)thio]-N-hydroxybutanamide (100% expansion, n=36) (H),methyl-4-(phenylthio)butanoate (89% expansion) (I), methyl4[(4-methoxyphenyl)thio]butanoate (91% expansion) (J), methyl4-[(4-methylphenyl)thio]butanoate (100% expansion) (K), methyl4-[(4-fluorophenyl)thio]butanoate (100% expansion) (L), propyl4-(phenylthio)butanoate (89% expansion) (M), and butan-2-yl4-(phenylthio)butanoate (89% expansion) (N).

FIG. 28. PTBA analogs exhibiting efficacy at 800 nM. (A through F) Insitu hybridization for lhx1a expression in 10-somite embryos treatedfrom 2 hpf with 0.5% DMSO (A) or 800 nM of the following compounds:methyl 4-[(methylphenyl)thio]butanoate (49% expansion) (B), methyl4-[(methoxyphenyl)thio]butanoate (57% expansion) (C),4-[(4-fluorophenyl)thio]-N-hydroxybutanamide (60% expansion) (D),N-hydroxy-4[(methylphenyl)thio]butanamide (61% expansion) (E), andmethyl 4-[(4-fluorophenyl)thio]butanoate (64% expansion, n=36) (F).

FIG. 29. PTBA analogs exhibiting efficacy at or below 400 nM. (A throughF) In situ hybridization for lhx1a expression in 10-somite embryostreated from 2 hpf with 0.5% DMSO (A) or concentrations of the followingcompounds as indicated: 400 nM4-[(4-fluorophenyl)thio]-N-hydroxybutanamide (35% expansion) (B), 400 nMmethyl 4-[(methoxyphenyl)thio]butanoate (29% expansion) (C), 200 nMmethyl 4-[(4-fluorophenyl)thio]butanoate (26% expansion) (D), 400 nMapicidin (22% expansion) (E), and 100 nM TSA (97% expansion) (F).

FIG. 30. Structural PTBA analogs exhibit low toxicity in culturedpodocytes. Podocytes were treated for 72 hours with 30 μM, 10 μM, 1 μM,300 nM, 100 nM, 30 nM, 10 nM, or 3 nM of each compound or a DMSOcontrol. Viability was assessed using Cell Titer-Blue. Viability wascalculated as a percentage of the DMSO control, which was considered100% viability. Data represent the average results of three independentexperiments using duplicate wells for each condition.

FIG. 31. HDACis enhance RA signaling. (A) In the absence of RA, RAR/RXRdimers recruit a corepressor complex (CoR) containing an HDAC. The HDACdeacetylates nucleosomes, causing chromatin condensation and preventingtranscription. (B) In the presence of RA, the RAR/RXR dimer undergoes aconformational change, removing the HDAC from close association withnucleosomes. This allows coactivators such as a histoneacetyltransferase (HAT) to acetylate the nucleosomes, which decondensesthe chromatin and permits transcription. (C) HDAC inhibitors, such asPTBA, have been hypothesized to decrease the required concentration ofRA necessary to trigger the RAR/RXR conformational switch. Figureadapted from Menegola and coworkers (Menegola, E., et al. Inhibition ofhistone deacetylase as a new mechanism of teratogenesis. Birth DefectsRes C Embryo Today 78, 345-353, (2006)).

FIG. 32. Treatment with MPTB increases the rate of renal recovery inmice following acute kidney injury. Female PTC-DTR mice were injectedwith 0.1 μg/kg DT on day 0. Beginning on day 1, mice were treated withdaily injections of 1% DMSO or 3.4 mg/kg MPTB (n=5 per group). Bloodurea nitrogen (BUN), a biomarker of nitrogenous wastes, was determinedevery two days following DT injection. Error bars are the standard errorof the mean. Asterisk represents a significant difference in BUNconcentration (p<0.002) as determined by t-test.

FIG. 33. Binding of TSA to human HDAC7 as determined by X-raycrystallography. (A) TSA co-crystallized with HDAC7 (ribbon model). (B)TSA co-crystallized with HDAC7 (space-filling model). (C) Magnificationof (B), showing coordination of the hydroxamic motif of TSA with thecatalytic site Zn²⁺.

FIG. 34. HDAC classes inhibited by carboxylic and hydroxamic acidHDACis. Carboxylic acid HDACis: butanoic acid, valproic acid, PBA, andTSA. Hydroxamic acid HDACis: TSA and SAHA. Class I, II, and IV HDACsutilize Zn2+-dependent catalysis, while the Class III sirtuins employ anNAD+-dependent mechanism.

FIG. 35. Evolutionary relationship between HDAC1 orthologs in selectedvertebrates. Cladogram generated using TreeView software (ver. 1.6.6)and HDAC1 RefSeq protein sequences for each species. Percentage denotespercent sequence identity with the human isoform. Scale bar representsnucleotide substitutions per site.

FIG. 36. Image based high-content screen. Tg(cad17:EGFP) zebrafishembryos arrayed in 96-well plates and the size of the fluorescentlylabeled kidney field quantified fish treated with the PTBA analogue,m4PTB. A/C shows GFP images in larvae at 56 hours; B/D showcorresponding CNT image analysis. Bar graph shows quantification of theidentified renal field (red).

FIG. 37. Renal histone acetylation after treatment with m4PTB. CD1 miceinjected S/C with the indicated doses of TSA or m4PTB, were sacrificedafter 24 hours, and Ac-H3 K9 evaluated in kidney nuclear extracts byWestern blot. Total H3 provides control for histone loading.

FIG. 38. Mouse model of AKI. Diphtheria toxin (DT) injected at day 0 inwild type and PTC-DTR mice (male). A-D, Renal histology at differenttime points after injection of 1 μg/kg DT. Trichrome blue stain showingcollagen deposition after 4 weeks (D). (E) BUN time course afterdifferent doses of DT in wild type and PTC-DTR mice. Mean+/−SD BUNmeasurements (6 mice per group). T-test, p<0.01*vs. wild type control, +vs. 0.1 μg/kg DT.

FIGS. 39A-39B. Effect of post-treatment with m4PTB on AKI. FemalePTC-DTR mice injected with 0.1 μg/kg DT injected and treated 24 hrslater with daily injections of 3.4 mg/kg m4PTB or DMSO control. (n=5 pergroup). (A) Renal function. Mean+/−SEM BUN, ANOVA, p<0.05, *DMSO vs.m4PTB; (B) Renal fibrosis. PAS and Trichrome staining of kidneysillustrating areas of tubulointerstitial fibrosis and tubular atrophy.Quantification of tubulointerstitial injury score (0-5, blindedanalysis). Mean+/−SEM, T-Test p<0.002, *DMSO (vehicle) vs. m4PTB.

FIGS. 40A-40B. Graphs showing EC₅₀s for PTBA (A) and m4PTB (B) done viaCNT analysis.

FIG. 41. Graph showing the results of an AKI model using HgCl₂ inducedinjury.

FIG. 42. Photomicrographs showing the results of an AKI model usingfolic acid-induced injury.

FIGS. 43A-43B. Graph showing the results of an AKI model usinggentamycin-induced injury (A) and table providing day results (B).

FIG. 44. Graph showing proliferation of renal cells examined post AKIfor m4PTB in zebrafish embryos.

DETAILED DESCRIPTION

The use of numerical values in the various ranges specified in thisapplication, unless expressly indicated otherwise, are stated asapproximations as though the minimum and maximum values within thestated ranges are both preceded by the word “about”. In this manner,slight variations above and below the stated ranges can be used toachieve substantially the same results as values within the ranges.Also, unless indicated otherwise, the disclosure of these ranges isintended as a continuous range including every value between the minimumand maximum values. For definitions provided herein, those definitionsrefer to word forms, cognates and grammatical variants of those words orphrases. “Comprising” and like terms are open-ended. The terms “a” and“an” refer to one or more.

As used herein, the term “patient” refers to members of the animalkingdom including but not limited to human beings and implies norelationship between a doctor or veterinarian and a patient.

Provided herein are compounds useful for improving kidney function,inhibiting a histone deacylase in a cell, expand renal progenitor cellsand/or stimulating kidney repair in cells in vitro, ex vivo or in vivo(in a patient). Compositions also are provided for delivery of thecompounds to a patient. Also provided are methods for improving kidneyfunction, inhibiting a histone deacylase in a cell, expand renalprogenitor cells and/or stimulating kidney repair in cells in vitro, exvivo or in vivo (in a patient) comprising contacting the cells with, oradministering to a patient and amount of one or more of the compoundseffective to improve kidney function in a patient, inhibit a histonedeacylase in a cell, expand renal progenitor cells and/or stimulatekidney repair in cells. Therefore provided are in vitro (including exvivo) or in vivo (in a patient) methods. Efficacy of the compounds isdemonstrated below.

The lead compound, PTBA, is unique in that it has relatively lowtoxicity when compared with several other established HDACi in zebrafishtoxicity assays (see below), and a unique thioester moiety in theposition of the connecting unit, a site that is typically occupied by anamide bond and has recently gained interest as a target for new drugdesign efforts. These findings have great potential because theyidentify for the first time a class of compounds that are non-toxic andaccelerate renal recovery by enhancing the innate potential of renaltubular epithelium to regenerate following injury. By validating the useof this screening strategy, our preliminary studies also indicate thatthis strategy can be used to identify chemical modifications andsynergistic interactions that will enhance the regenerative capacity oflead compounds both in the zebrafish embryo and subsequently in mousemodels of AKI. Thus the examples presented below provide a uniqueopportunity to develop a panel of compounds that could have a realimpact on the clinical outcome of a large number of patients presentingwith AKI, particularly those with severe AKI in which potentially themajority of naturally occurring self-renewing epithelial cells have beendepleted. In FIG. 1, exemplary HDACi analogues of PTBA are shown, withPTBA in the center. Potential HDACi analogue families to test include:esterified compounds; esterified compounds with bulk added to the phenolring for increased Log P; hydroxamic compounds with bi-dentate zincchelator; hydroxamic compounds with bulk added to the phenol ring forincreased Log P; and, other proven HDACi, such as SAHA and TSA.

According to one non-limiting embodiment, a compound is provided havingthe formula:

in which

-   -   R is S, S(O), S(O)₂ or NH,    -   R1 is —NH—R4 where R4 is OH, aminophenyl, hydroxyphenyl, C₁₋₄        alkyl hydroxyphenyl or phenyl hydroxyphenyl, or —O—R5 where R5        is H or C₁₋₄ alkyl,    -   R2 is phenyl; substituted phenyl;

-   -    where R5 is halo; C₁₋₄ alkyl, methyl; methoxy, C₁₋₄ alkoxy;        naphthyl; 1H-1,3-benzodiazol-2-yl; 1,3-benzothiazol-2-yl;        pyrimidinyl, 1-methyl-1H-1,3benzodiazol-2-yl; pyridyl;        methoxyphenyl; or methylthiophenyl, and R3 is from 0 to 5        methylene groups ((—CH₂-)₀₋₅) and 0 or 1 phenylene wherein at        least one methylene or phenylene is present,        or a pharmaceutically acceptable salt thereof, other than        4-(phenylthio)butanoic acid (PTBA, also        4-(phenylsulfanyl)butanoic acid). In one example, R is S. R3 is        an aliphatic alkylene, such as —CH₂—CH₂—CH₂—, —CH₂—CH₂—CH₂—CH₂—        or —CH₂—CH₂—CH₂—CH₂—CH₂— that may include one phenylene group

for example, R3 can be;

According to certain examples, R2 is phenyl, 4-fluorophenyl,4-methoxyphenyl or 4-methylphenyl and/or R3 is —CH₂—CH₂—CH₂— or—CH₂—CH₂—CH₂—CH₂—CH₂—. In other examples, R1 is —NH—OH,—NH-2-aminophenyl, —NH-2-hydroxyphenyl or —O—CH₃. Specific examples ofuseful compounds include: UPHD-00029; UPHD-00028; UPHD-00034;UPHD-00051; UPHD-00067; UPHD-00025; UPHD-00030; UPHD-00022; UPHD-00047;UPHD-00048; UPHD-00049; UPHD-00053; UPHD-00077; or pharmaceuticallyacceptable salts thereof. Structures of non-limiting compounds describedherein and useful in the methods described herein are provided in Table1.

TABLE 1 Compound structure ID MW EF LogP Name

UPHD-00020 196.27 C₁₀H₁₂O₂S 2.5 4-(phenylthio) butanoic acid (PTBA)

UPHD-00023 (VNK-I-276) 211.28 C₁₀H₁₃NO₂S 1.69 N-hydroxy-4(phenylsulfanyl) butanamide

UPHD-00029 (VNK-I-294) 229.27 C₁₀H₁₂FNO₂S 1.83 4-[(4- fluorophenyl)sulfanyl]-N- hydroxy- butanamide

UPHD-00028 (VNK-I-305) 225.31 C₁₁H₁₅NO₂S 2.2 N-hydroxy- 4-[(4-methylphenyl) sulfanyl] butanamide

UPHD-00027 (VNK-I-287) 245.73 C₁₀H₁₂ClNO₂S 2.3 4-[(4- chlorophenyl)sulfanyl]-N- hydroxy- butanamide

UPHD-00097 258.2909 (Exact = 258.0562) C₁₁H₁₄O₅S 0.81 4-[(4- methoxy-benzene)sulfonyl] butanoic acid

UPHD-00035 225.3073 C₁₁H₁₅NO₂S 2.14 N-hydroxy-5- (phenylsulfanyl)pentanamide

UPHD-00034 243.3 C₁₁H₁₄FNO₂S 2.28 5-[(4- fluorophenyl) sulfanyl]-N-hydroxy- pentanamide

UPHD-00050 239.3339 C₁₂H₁₇NO₂S 2.72 N-hydroxy-6- (phenylsulfanyl)hexanamide

UPHD-00051 257.3243 C₁₂H₁₆FNO₂S 3.07 6-[(4- fluorophenyl) sulfanyl]-N-hydroxy- hexanamide

UPHD-00119 257.3243 (Exact = 257.0886) C₁₂H₁₆FNO₂S 2.72 6-[(3-fluorophenyl) sulfanyl]-N- hydroxy- hexanamide

UPHD-00118 257.3243 (Exact = 257.0886) C₁₂H₁₆FNO₂S 2.72 6-[(2-fluorophenyl) sulfanyl]-N- hydroxy- hexanamide

UPHD-00143 273.7789 (Exact = 273.059) C₁₂H₁₆ClNO₂S 6-[(4- fluorophenyl)sulfanyl]-N- hydroxy- hexanamide

UPHD-00130 269.3599 (Exact = 269.1086) C₁₃H₁₉NO₃S 2.42 N-hydroxy-6-[(4-methoxy- phenyl)sulfanyl] hexanamide

UPHD-00131 269.3599 (Exact = 269.1086) C₁₃H₁₉NO₃S 2.42 N-hydroxy-6-[(3-methoxy- phenyl)sulfanyl] hexanamide

UPHD-00132 269.3599 (Exact = 269.1086) C₁₃H₁₉NO₃S 2.42 N-hydroxy-6-[(2-methoxy- phenyl)sulfanyl] hexanamide

UPHD-00041 253.3605 C₁₃H₁₉NO₂S 3.02 N-hydroxy-7- (phenylsulfanyl)heptanamide

UPHD-00042 271.3509 C₁₃H₁₈FNO₂S 3.17 7-[(4-fluoro- phenyl)sulfanyl]-N-hydroxy- heptanamide

UPHD-00052 259.3235 C₁₄H₁₃NO₂S 3.07 N-hydroxy-4- [(phenylsulfanyl)methyl]benzamide

UPHD-00067 291.3406 (Exact = 291.0729) C₁₅H₁₄FNO₂S 3.5 4-{2[(4-fluoro-phenyl)sulfanyl] ethyl}-N- hydroxy- benzamide

UPHD-00061 273.3501 (Exact = 273.0823) C₁₅H₁₅NO₂S 3.36 N-hydroxy-4-[2-(phenyl- sulfanyl)ethyl] benzamide

UPHD-00074 241.31 (Exact = 241.0885) C₁₀H₁₅N₃O₂S 1.34 N-hydroxy-6-(pyrmidin-2- ylsulfanyl) hexanamide

UPHD-00059 289.3926 (Exact = 289.1136) C₁₆H₁₉NO₂S 3.57 N-hydroxy-6-(naphthalen-2- ylsulfanyl) hexanamide

UPHD-00069 321.3914 (Exact = 321.1035) C₁₆H₁₉NO₄S 2.04 N-hydroxy-6-(naphthalene-2- sulfonyl) hexanamide

UPHD-00070 296.4083 (Exact = 296.0653) C₁₃H₁₆N₂O₂S2 3.4 6-(1,3-benzothiazol- 2-ylsulfanyl)- N-hydroxy- hexanamide

UPHD-00085 293.3846 (Exact = 293.1198) C₁₄H₁₉N₃O₂S 2.77 N-hydroxy-6-[(1-methyl-1H- 1,3benzodiazol- 2-yl)sulfanyl] hexanamide

UPHD-00024 238 C₁₃H₁₈O₂S 3.53 propyl 4- (phenylsulfanyl) butanoate

UPHD-00025 210.29 C₁₁H₁₄O₂S 2.65 methyl 4- (phenylsulfanyl) butanoate(m4PTB)

UPHD-00030 (VNK-I-291) 228.28 C₁₁H₁₃FO₂S 2.79 methyl 4-[(4-fluorophenyl) sulfanyl]butanoate

UPHD-00021 (VNK-I-284) 244.74 C₁₁H₁₃ClO₂S 3.25 methyl 4-[(4-chlorophenyl) sulfanyl]butanoate

UPHD-00026 (VNK-I-285) 224.32 C₁₂H₁₆O₂S 3.16 methyl 4-[(4- methylphenyl)sulfanyl]butanoate

UPHD-00022 (VNK-I-286) 240.32 C₁₂H₁₆O₃S 2.49 methyl 4-[(4-methoxyphenyl) sulfanyl]butanoate

UPHD-00033 224.3192 C₁₂H₁₆O₂S 3.09 methyl 5- (phenylsulfanyl) pentanoate

UPHD-00032 242.3097 C₁₂H₁₅FO₂S 3.23 methyl 5-[(4- fluorophenyl)sulfanyl]pentanoate

UPHD-00047 238.3458 C₁₃H₁₈O₂S 3.68 methyl 6- (phenylsulfanyl) hexanoate

UPHD-00048 256.3363 C₁₃H₁₇FO₂S 3.54 methyl 6-[(4- fluorophenyl)sulfanyl]hexanoate

UPHD-00088 256.3363 (Exact = 256.0933) C₁₃H₁₇FO₂S 3.68 methyl 6-[(3-fluorophenyl) sulfanyl]hexanoate

UPHD-00089 256.3363 (Exact = 256.0933) C₁₃H₁₇FO₂S 3.68 methyl 6-[(2-fluorophenyl) sulfanyl]hexanoate

UPHD-00111 272.7909 (Exact = 272.0638) C₁₃H₁₇ClO₂S 4.14 methyl 6-[(4-chlorophenyl) sulfanyl]hexanoate

UPHD-00113 272.7909 (Exact = 272.0638) C₁₃H₁₇ClO₂S 4.14 methyl 6-[(3-chlorophenyl) sulfanyl]hexanoate

UPHD-00112 272.7909 (Exact = 272.0638) C₁₃H₁₇ClO₂S 4.14 methyl 6-[(2-chlorophenyl) sulfanyl]hexanoate

UPHD-00144 254.3452 (Exact = 254.0977) C₁₃H₁₈O₃S methyl 6-[(4-hydroxyphenyl) sulfanyl]hexanoate

UPHD-00106 254.3452 (Exact = 254.0977) C₁₃H₁₈O₃S 1.9 methyl 6-(phenylsulfinyl) hexanoate

UPHD-00107 272.3357 (Exact = 272.0882) C₁₃H₁₇FO₃S 2.04 methyl 6-[(4-fluoro- phenyl)sulfinyl] hexanoate

UPHD-00091 270.3446 (Exact = 270.0926) C₁₃H₁₈O₄S 2 methyl 6-(benzenesulfonyl) hexanoate

UPHD-00092 288.3351 (Exact = 288.0832 C₁₃H₁₇FO₄S 2.15 methyl 6-[(4-fluorobenzene) sulfonyl]hexanoate

UPHD-00090 268.3718 (Exact = 268.1133) C₁₄H₂₀O₃S 3.38 methyl 6-[(4-methoxyphenyl) sulfanyl]hexanoate

UPHD-00100 268.3718 (Exact = 268.1133) C₁₄H₂₀O₃S 3.38 methyl 6-[(3-methoxyphenyl) sulfanyl]hexanoate

UPHD-00101 268.3718 (Exact = 268.1133) C₁₄H₂₀O₃S 3.38 methyl 6-[(2-methoxyphenyl) sulfanyl]hexanoate

UPHD-00135 296.3819 (Exact = 296.1082) C₁₅H₂₀O₄S 3.54 methyl 4-[(6-methoxy-6- oxohexyl)sulfanyl] benzoate

UPHD-00040 252.3724 C₁₄H₂₀O₂S 3.98 methyl 7-(phenyl- sulfanyl)heptanoate

UPHD-00039 270.3629 C₁₄H₁₉FO₂S 4.12 methyl 7-[(4- fluorphenyl)sulfanyl]heptanoate

UPHD-00045 258.3355 C₁₅H₁₄O₂S 4.23 methyl 4[(phenyl- sulfanyl)methyl]benzoate

UPHD-00046 276.3259 C₁₅H₁₃FO₂S 4.37 methyl 4-{[(4- fluorophenyl)sulfanyl]methyl} benzoate

UPHD-00057 288.4045 (Exact = 288.1184) C₁₇H₂₀O₂S 4.53 methyl 6-(naphthalen-2- ylsulfanyl)hexanoate

UPHD-00063 320.4033 (Exact = 320.1082) C₁₇H₂₀O₄S 2.99 methyl 6-(naphthalen-2- ylsulfonyl)hexanoate

UPHD-00062 295.4203 (Exact = 295.0701) C₁₄H₁₇NO₂S₂ 4.36 methyl 6-(1,3-benzothiazol-2- ylsulfanyl) hexanoate)

UPHD-00076 278.3699 (Exact = 278.1089) C₁₄H₁₈N₂O₂S 3.51 methyl 6-(1H-1,3benzodiazol-2- ylsulfanyl)hexanoate

UPHD-00058 240.3219 (Exact = 240.0932) C₁₁H₁₆N₂O₂S 2.29 methyl 6-(pyrimidin-2- ylsulfanyl)hexanoate

UPHD-00075 292.3965 (Exact = 292.1245) C₁₅H₂₀N₂O₂S 3.73 methyl 6-[(1-methyl-1H-1,3- benzodiazol-2- yl)sulfanyl] hexanoate

UPHD-00065 239.3339 (Exact = 239.098) C₁₂H₁₇NO₂S 2.91 methyl 6-(pyridin-2-ylsulfanyl) hexanoate

UPHD-00142 284.4374 (Exact = 284.0905) C₁₄H₂₀O₂S₂ methyl 6-[(4-(methylsulfanyl) phenyl)sulfanyl] hexanoate

UPHD-00094 256.318 (Exact = 256.0769) C₁₂H₁₆O₄S 0.85 methyl 4-[4-methyoxy- benzene)sulfinyl] butanoate

UPHD-00129 344.1558 (Exact = 344.4711) C₁₉H₂₄N₂O₂S 3.84 N-(2-amino-phenyl)-6-[(2- methoxyphenyl) sulfanyl]hexanamide

UPHD-00093 272.3174 (Exact = 272.0718) C₁₂H₁₆O₅S 0.96 methyl 4-[(4-methoxybenzene) sulfonyl]butanoate

UPHD-00036 286.3919 C₁₆H₁₈N₂OS 3.11 N-(2-aminophenyl)-4-(phenylsulfanyl) butanamide

UPHD-00038 300.4185 C₁₇H₂₀N₂OS 3.55 N-(2-aminophenyl)-5-(phenylsulfanyl) pentanamide

UPHD-00037 318.409 C₁₇H₁₉FN₂OS 3.69 N-(2-amino- phenyl)-5-[(4-fluorophenyl) sulfanyl]pentanamide

UPHD-00049 314.4451 (Exact = 314.1453) C₁₈H₂₂N₂OS 4 N-(2-aminophenyl)-6-(phenylsulfanyl) hexanamide

UPHD-00053 332.4355 (Exact = 332.1359) C₁₈H₂₁FN₂OS 4.14N-(2-aminophenyl)- 6-[(4-fluorphenyl) sulfanyl]hexanamide

UPHD-00136 332.4355 (Exact = 332.1359) C₁₈H₂₁FN₂OS 4.14 N-(2-amino-phenyl)-6-[(3- fluorophenyl)sulfanyl] hexanamide

UPHD-00122 332.4355 (Exact = 332.1359) C₁₈H₂₁FN₂OS 4.14 N-(2-amino-phenyl)-6-[(2- fluorophenyl)sulfanyl] hexanamide

UPHD-00125 344.4711 (Exact = 344.1558) C₁₉H₂₄N₂O₂S 3.84 N-(2-amino-phenyl)-6-[(4- methoxy- phenyl)sulfanyl] hexanamide

UPHD-00126 344.4711 (Exact = 344.1558) C₁₉H₂₄N₂O₂S 3.84 N-(2-amino-phenyl)-6-[(3- methoxy- phenyl)sulfanyl] hexanamide

UPHD-00128 348.8901 (Exact = 348.1063) C₁₈H₂₁ClN₂OS 4.6 N-(2-amino-phenyl)-6-[(4- chloro- phenyl)sulfanyl] hexanamide

UPHD-00077 333.4203 (Exact = 333.1199) C₁₈H₂₀FNO₂S 4.66 6-[(4-fluoro-phenyl)sulfanyl]- N-(2-hydroxy- phenyl)hexanamide

UPHD-00115 333.4203 (Exact = 333.1199) C₁₈H₂₀FNO₂S 4.66 6-[(3-fluoro-phenyl)sulfanyl]- N-(2-hydroxy- phenyl)hexanamide

UPHD-00114 333.4203 (Exact = 333.1199) C₁₈H₂₀FNO₂S 4.66 6-[(2-fluoro-phenyl)sulfanyl]- N-(2-hydroxy- phenyl)hexanamide

UPHD-00123 345.4558 (Exact = 345.1399) C₁₉H₂₃NO₃S 4.36 N-(2-hydroxyphenyl)- 6-[(3- methoxyphenyl) sulfanyl]hexanamide

UPHD-00121 345.4558 (Exact = 345.1399) C₁₉H₂₃NO₃S 4.36 N-(2-hydroxy-phenyl)-6-[(2- methoxyphenyl) sulfanyl]hexanamide

UPHD-00124 345.4558 (Exact = 345.1399) C₁₉H₂₃NO₃S 4.36 N-(2-hydroxy-phenyl)-6-[(4- methoxyphenyl) sulfanyl]hexanamide

UPHD-00138 349.8749 (Exact = 349.0903) C₁₈H₂₀ClNO₂S 5.13 6-[(3-chloro-phenyl)sulfanyl]- N-(2-hydroxy- phenyl)hexanamide

UPHD-00127 349.8749 (Exact = 349.0903) C₁₈H₂₀ClNO₂S 5.13 6-[(4-chloro-phenyl)sulfanyl]- N-(2-hydroxy- phenyl)hexanamide

UPHD-00140 331.4292 (Exact = 331.1242) C₁₈H₂₁NO₃S 2.88 6-(benzene-sulfinyl)-N-(2- hydroxyphenyl) hexanamide

UPHD-00141 349.4197 (Exact = 349.1148) C₁₈H₂₀FNO₃S 6-(4-fluoro-benzenesulfinyl)- N-(2-hydroxy- phenyl)hexanamide

UPHD-00044 328.4717 C₁₉H₂₄N₂OS 4.44 N-(2-amino- phenyl)-7-(phenylsulfanyl) heptanamide

UPHD-00043 346.4621 C₁₉H₂₃FN₂OS 4.58 N-(2-amino- phenyl)-7-[(4- fluoro-phenyl)sulfanyl] heptanamide

UPHD-00073 315.4331 (Exact = 315.1405) C₁₇H₂₁N₃OS 3.37 N-(2-amino-phenyl)-6-(pyridin- 2-ylsulfanyl) hexanamide

UPHD-00072 316.4212 (Exact = 316.1358) C₁₆H₂₀N₄OS 2.75 N-(2-amino-phenyl)-6- (pyrimidin-2- ylsulfanyl) hexanamide

UPHD-00086 317.406 (Exact = 317.1198) C₁₆H₁₉N₃O₂S 3.28 N-(2-hydroxy-phenyl)-6- (pyrimidin- 2-ylsulfanyl) hexanamide

UPHD-00087 373.5123 (Exact = 373.1824) C₂₀H₂₇N₃O₂S 4.82 N-(5-tert-butyl-2-hydroxy- phenyl)-6- (pyrimidin-2- ylsulfanyl) hexanamide

UPHD-00060 364.5038 (Exact = 364.1609) C₂₂H₂₄N₂OS 4.99 N-(2-amino-phenyl)-6- (naphthalen-2- ylsulfanyl) hexanamide

UPHD-00066 396.5026 (Exact = 396.1508) C₂₂H₂₄N₂O₃S 3.45 N-(2-amino-phenyl)-6- (naphthalene-2- sulfonyl) hexanamide

UPHD-00071 371.5195 (Exact = 371.1126) C₁₉H₂₁N₃OS₂ 4.82 N-(2-amino-phenyl)-6-(1,3- benzothiazol-2- ylsulfanyl) hexanamide

UPHD-00081 368.4958 (Exact = 368.1671) C₂₀H₂₄N₄OS 4.19 N-(2-amino-phenyl)-6- [(1-methyl-1H- 1,3-benzodiazol- 2-yl)sulfanyl] hexanamide

UPHD-00095 355.4539 (Exact = 355.1354) C₁₉H₂₁N₃O₂S 4.49 6-(1H-1,3-benzodiazol-2- ylsulfanyl)-N-(2- hydroxyphenyl) hexanamide

UPHD-00054 334.4347 (Exact = 334.114) C₂₀H₁₈N₂OS 4.49 N-(2-amino-phenyl)-4- [(phenylsulfanyl) methyl]benzamide

UPHD-00055 352.4252 (Exact = 352.1046) C₂₀H₁₇FN₂OS 4.63 N-(2-amino-phenyl)-4-([(4- fluorophenyl) sulfanyl]methyl) benzamide

UPHD-00064 348.4612 (Exact = 348.1296) C₂₁H₂₀N₂OS 4.77 N-(2-amino-phenyl)-4-[2- (phenylsulfanyl) ethyl]benzamide

UPHD-00068 366.4518 (Exact = 366.1202) C₂₁H₁₉FN₂OS 4.92 N-(2-amino-phenyl)-4-{2-[(4- fluoro- phenyl)sulfanyl] ethyl}benzamide

UPHD-00104 347.4286 (Exact = 347.1191) C₁₈H₂₁O₄S 2.99 N-(2-hydroxy-phenyl)-6- (phenylsulfonyl) hexanamide

UPHD-00105 365.4191 (Exact = 365.1097) C₁₈H₂₀FNO₄S 3.13 6-[(4-fluoro-phenyl)sulfonyl]- N-(2-hydroxy- phenyl)hexanamide

UPHD-00084 347.4469 (Exact = 347.1355) C₁₉H₂₂FNO₂S 5.18 6-[(4-fluoro-phenyl)sulfanyl]- N-(2-hydroxy- 5-methylphenyl) hexanamide

UPHD-00083 389.5266 (Exact = 389.1825) C₂₂H₂₈FNO₂S 6.21 N-(5-tert-butyl-2-hydroxy- phenyl)-6-[(4- fluorophenyl) sulfanyl]hexanamide

UPHD-00082 406.5163 (Exact = 409.1512) C₂₄H₂₄FNO₂S 6.31 6-[(4-fluoro-phenyl)sulfanyl]- N-(2-hydroxy- 5-phenylphenyl) hexanamide

UPHD-00056 258.3355 (Exact = 258.0715) C₁₅H₁₄O₂S 4.52 ethyl 4-(phenyl-sulfanyl)benzoate

UPHD-00096 300.4152 (Exact = 300.1184) C₁₈H₂₀O₂S 5.32 ethyl 4-[3-(phenylsulfanyl) propyl]benzoate

VNK-I-154 (1-methyl- propyl)-4- (phenylsulfanyl) butanoate

VNK-I-157 n-butyl-4- (phenylsulfanyl) butanoate

VNK-I-259 4-[phenyl- sulfanyl]-N- methylbutanamide

VNK-I-300 4-(phenyl- amino)butanoic acid

VNK-I-290 methyl-4-(4- (bromophenyl) sulfanyl)butanoate

VNK-I-289 4-[(4-methoxy- phenyl)sulfanyl]-N- hydroxybutanamide

VNK-I-292 4-[(4-bromo- phenyl)sulfanyl]-N- hydroxybutanamide

VNK-I-298 methyl 4- (phenylamino) butanoate

Pharmaceutically acceptable salts of any of the compounds describedherein also may be used in the methods described herein.Pharmaceutically acceptable salt forms of the compounds described hereinmay be prepared by conventional methods known in the pharmaceuticalarts, and include as a class veterinarily acceptable salts. For exampleand without limitation, where a compound comprises a carboxylic acidgroup, a suitable salt thereof may be formed by reacting the compoundwith an appropriate base to provide the corresponding base additionsalt. Non-limiting examples include: alkali metal hydroxides, such aspotassium hydroxide, sodium hydroxide and lithium hydroxide; alkalineearth metal hydroxides, such as barium hydroxide and calcium hydroxide;alkali metal alkoxides, such as potassium ethanolate and sodiumpropanolate; and various organic bases such as piperidine,diethanolamine, and N-methylglutamine.

Acid and base addition salts may be prepared by contacting the free baseform with a sufficient amount of a desired acid or base to produce thesalt in a manner known in the art. The free base may be regenerated bycontacting the salt form with a base or acid (depending on the nature ofthe salt) and isolating the free base. The free base forms differ fromtheir respective salt forms somewhat in certain physical properties suchas solubility in polar solvents, but otherwise the salts are equivalentto their respective free base forms for purposes described herein.

Compounds comprising basic nitrogen-containing groups may be quaternizedwith such agents as C₁₋₄ alkyl halides, such as methyl, ethyl,iso-propyl and tert-butyl chlorides, bromides and iodides; C₁₋₄ alkylsulfate such as dimethyl, diethyl and diamyl sulfates; C₁₀₋₁₈ alkylhalides, such as decyl, dodecyl, lauryl, myristyl and stearyl chlorides,bromides and iodides; and aryl-C₁₋₄ alkyl halides, such as benzylchloride and phenethyl bromide. Such salts permit the preparation ofboth water-soluble and oil-soluble compounds.

Non-limiting examples of pharmaceutically-acceptable base salts include:aluminum, ammonium, calcium, copper, ferric, ferrous, lithium,magnesium, manganic, manganous, potassium, sodium, and zinc salts. Saltsderived from pharmaceutically acceptable organic non-toxic basesinclude, without limitation: salts of primary, secondary, and tertiaryamines, substituted amines including naturally occurring substitutedamines, cyclic amines, and basic ion exchange resins, such as arginine,betaine, caffeine, chloroprocaine, choline, N,N′-dibenzylethylenediamine(benzathine), dicyclohexylamine, diethanolamine, diethylamine,2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine,ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine,glucosamine, histidine, hydrabamine, iso-propylamine, lidocaine, lysine,meglumine, N-methyl-D-glucamine, morpholine, piperazine, piperidine,polyamine resins, procaine, purines, theobromine, triethanolamine,triethylamine, trimethylamine, tripropylamine, andtris-(hydroxymethyl)-methylamine (tromethamine).

Acid addition salts may be prepared by treating a compound withpharmaceutically acceptable organic and inorganic acids, including,without limitation: hydrohalides, such as hydrochloride, hydrobromide,hydroiodide; other mineral acids and their corresponding salts such assulfates, nitrates, and phosphates; alkyl- and mono-arylsulfonates, suchas ethanesulfonate, toluenesulfonate, and benzenesulfonate; and otherorganic acids and their corresponding salts, such as acetate, tartrate,maleate, succinate, citrate, benzoate, salicylate, and ascorbate.

Non-limiting examples of pharmaceutically-acceptable acid salts include:acetate, adipate, alginate, arginate, aspartate, benzoate, besylate(benzenesulfonate), bisulfate, bisulfite, bromide, butyrate, camphorate,camphorsulfonate, caprylate, chloride, chlorobenzoate, citrate,cyclopentanepropionate, digluconate, dihydrogenphosphate,dinitrobenzoate, dodecylsulfate, ethanesulfonate, fumarate, galacterate,galacturonate, glucoheptanoate, gluconate, glutamate, glycerophosphate,hemisuccinate, hemisulfate, heptanoate, hexanoate, hippurate,hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate,iodide, isethionate, iso-butyrate, lactate, lactobionate, malate,maleate, malonate, mandelate, metaphosphate, methanesulfonate,methylbenzoate, monohydrogenphosphate, 2-naphthalenesulfonate,nicotinate, nitrate, oxalate, oleate, pamoate, pectinate, persulfate,phenylacetate, 3-phenylpropionate, phosphate, phosphonate, andphthalate.

Multiple salts forms are also considered to bepharmaceutically-acceptable salts. Common, non-limiting examples ofmultiple salt forms include: bitartrate, diacetate, difumarate,dimeglumine, diphosphate, disodium, and trihydrochloride.

As such, “pharmaceutically acceptable salt” as used herein is intendedto mean an active ingredient (drug) comprising a salt form of anycompound as described herein. The salt form preferably confers to theimproved and/or desirable pharmacokinetic/pharmodynamic properties ofthe compounds described herein.

In use, any compound described herein, including pharmaceuticallyacceptable salts thereof, may be admixed with any pharmaceuticallyacceptable carrier or carriers, such as water, saline, physiologicalsalt solutions, Ringer's solution or any other carrier customarily usedfor administration of drugs to the subject in question (see, generally,Troy, DB, Editor, Remington: The Science and Practice of Pharmacy, 21stEd., Lippincott Williams & Wilkins (2005), pp. 745-849 for descriptionsof various compositions, solutions, and dosage forms useful foradministration of the described compounds, as well as methods of makingsuch compositions, solutions, and dosage forms).

According to one non-limiting example, the compounds described hereinare formulated into a composition with one or more pharmaceuticalvehicles or diluents for oral, intravenous or subcutaneousadministration. The composition can be formulated in a classical mannerusing solid or liquid vehicles, diluents and additives appropriate tothe desired mode of administration. Orally, the compounds can beadministered in the form of tablets, capsules, granules, powders and thelike.

In any case, as used herein, any agent or agents used for improvingkidney function, inhibiting a histone deacylase in a cell, expanding apopulation of renal progenitor cells and/or stimulating kidney repair incells in vitro, ex vivo or in vivo is administered in an amounteffective to improve kidney function, to inhibit a histone deacylase ina cell, to expand renal progenitor cells and/or to stimulate kidneyrepair in cells in vitro, ex vivo or in vivo, namely in an amount and ina dosage regimen effective to improve kidney function, inhibit a histonedeacylase in a cell, expand renal progenitor cells and/or stimulatekidney repair in cells in vitro, ex vivo or in vivo. According to onenon-limiting embodiment, an effective dose ranges from 0.05 to 200mg/kg/day, and in certain embodiments less than 100 mg/kg/day, includingany increment or range therebetween, including 0.1 mg/kg/day, 0.5mg/kg/day, 1 mg/kg/day, 5 mg/kg/day, 10 mg/kg/day, 20 mg/kg/day, 25mg/kg/day, 50 mg/kg/day, 75 mg/kg/day, 100 mg/kg/day, etc. However, foreach compound described herein, an effective dose or dose range isexpected to vary from that of other compounds described herein for anynumber of reasons, including the molecular weight of the compound,bioavailability in the dosage form, route of administration, specificactivity (e.g., EC₅₀), etc. In vitro (including ex vivo), thecomposition is used, for example, in culture medium. Exemplary andnon-limiting effective ranges range from 100 nM to 25 μM, 200 nM to 3μM, 200 nM to 1.5 μM, including all increments therebetween. Once again,the effective range and optimal concentration range depends on thespecific activity (e.g., EC₅₀) of the composition, as well as a varietyof other conditions. In any case, the effective range (e.g., thetherapeutic window) between the minimally-effective dose, and maximumtolerable dose in a subject can be determined empirically by a person ofskill in the art, with end points being determinable by in vitro and invivo assays, such as those described herein and/or are acceptable in thepharmaceutical and medical arts for obtaining such information regardingagents, such as histone deacetylase inhibitors. Different concentrationsof the agents described herein are expected to achieve similar results.The compounds can be administered orally one or more times daily, forexample two to four times daily, once every two, three, four, five ormore days, weekly, monthly, etc., including increments therebetween. Incertain delivery methods, it is possible to deliver the drugcontinuously, or substantially continuously as in the case of, forexample, intravenous or transdermal delivery routes. A person ofordinary skill in the pharmaceutical and medical arts will appreciatethat it will be a matter of design choice and/or optimization toidentify a suitable dosage regimen for improving kidney function,inhibiting a histone deacylase in a cell, expand renal progenitor cellsand/or stimulating kidney repair in cells in vitro, ex vivo or in vivo.

The compounds described herein may be administered in any manner that iseffective to improve kidney function, inhibit a histone deacylase in acell, expand a renal progenitor cell population and/or stimulate kidneyrepair in cells in vitro, ex vivo or in vivo. Examples of deliveryroutes include, without limitation: topical, for example, epicutaneous,inhalational, enema, ocular, otic and intranasal delivery; enteral, forexample, orally, by gastric feeding tube or swallowing, and rectally;and parenteral, such as, intravenous, intraarterial, intramuscular,intracardiac, subcutaneous, intraosseous, intradermal, intrathecal,intraperitoneal, transdermal, iontophoretic, transmucosal, epidural andintravitreal, with oral or intravenous approaches being preferred forimproving kidney function, inhibiting a histone deacylase in a cell,expanding a population of renal progenitor cells and/or stimulatingkidney repair in cells in vitro, ex vivo or in vivo.

Therapeutic/pharmaceutical compositions are prepared in accordance withacceptable pharmaceutical procedures, such as described in Remington:The science and Practice of Pharmacy, 21st edition, ed. Paul Beringer etal., Lippincott, Williams & Wilkins, Baltimore, Md. Easton, Pa. (2005)(see, e.g., Chapters 37, 39, 41, 42 and 45 for examples of powder,liquid, parenteral, intravenous and oral solid formulations and methodsof making such formulations).

Any of the compounds described herein may be compounded or otherwisemanufactured into a suitable composition for use, such as apharmaceutical dosage form or drug product in which the compound is anactive ingredient. According to one example, the drug product describedherein is an oral tablet, capsule, caplet, liquid-filled or gel-filledcapsule, etc. Compositions may comprise a pharmaceutically acceptablecarrier, or excipient. An excipient is an inactive substance used as acarrier for the active ingredients of a medication. Although “inactive,”excipients may facilitate and aid in increasing the delivery, stabilityor bioavailability of an active ingredient in a drug product.Non-limiting examples of useful excipients include: antiadherents,binders, rheology modifiers, coatings, disintegrants, emulsifiers, oils,buffers, salts, acids, bases, fillers, diluents, solvents, flavors,colorants, glidants, lubricants, preservatives, antioxidants, sorbents,vitamins, sweeteners, etc., as are available in thepharmaceutical/compounding arts.

According to one non-limiting embodiment, the compounds described hereinare complexed with a cyclodextrin. Cyclodextrins are compounds that havefound substantial recognition as excipients (e.g., as carriers,vehicles, etc.) in the pharmaceutical field, for example in oral andintravenous dosage forms. Cyclodextrins are able form non-covalentinclusion complexes and/or aggregates in solution with poorly solubledrugs, for example, BCS Class II and IV drugs (high or low intestinalpermeability, respectively, but low solubility in both instances).Cyclodextrins are cyclic oligosaccharides having a hydrophilic outersurface and a lipophilic central cavity. They consist of α-1,4-linkedα-D-glucopyranose units. Naturally-occurring cyclodextrins include α-,β- and γ-cyclodextrins, with 6, 7 and 8 glucopyranose units,respectively. The natural cyclodextrins can be used orally or topically,but natural β-cyclodextrin and γ-cyclodextrin cannot be usedparenterally. A number of cyclodextrin derivatives have been formulatedwith various usefulness in different administrative routes. Common,non-limiting examples of cyclodextrin derivatives includehydroxypropyl-β-cyclodextrin (e.g., 2-hydroxypropyl-β-cyclodextrin),hydroxypropyl-γ-cyclodextrin (e.g., 2-hydroxypropyl-γ-cyclodextrin),hydroxyethyl-β-cyclodextrin, randomly methylated β-cyclodextrin,methyl-β-cyclodextrin, dimethyl-β-cyclodextrin, permethylatedβ-cyclodextrin, sulfobutylether β-cyclodextrin (e.g., sodium salt),sulfobutyl-γ-cyclodextrin, branched cyclodextrin (e.g.,glucosyl-β-cyclodextrin or maltosyl-β-cyclodextrin, e.g.,6-O-maltosyl-β-cyclodextrin or glucosyl-β-cyclodextrin) andrandomly-acetylated amorphous-β-cyclodextrin. Cyclodextrins may becomplexed with a drug as inclusion complexes (included) in a solution ina 1:1 molar ratio, though increased or decreases relative amounts of thedrug or cyclodextrin may be used during formulation in order to drivethe reaction. Where the drug is aggregated instead of included withinthe cyclodextrin, an excess of cyclodextrin may be utilized. It shouldbe recognized that the inclusion or aggregation process can beoptimized, including manipulation of relative cyclodextrin-to-activeingredient ratios to obtain optimal solubility and bioavailability orother desirable features of the end-product.

A complete description of the state of the art of the uses ofcyclodextrins as pharmaceutical excipients is beyond the scope of thisdocument. To this end, see, Loftsson et al. “Self-Association ofCyclodextrins and Cyclodextrin Complexes” J. Pharm. Sci. 93(5):1091-1099(2004); Loftsson et al. “Cyclodextrins in Drug Delivery” Expert. Opin.Drug Deliv. 2:335-351 (2005); Brewster et al. “Cyclodextrins asPharmaceutical Solubilizers” Advanced Drug Delivery Reviews 59:645-666(2007); and Rasheed et al., “Cyclodextrins as Drug Carrier Molecule: Areview” Sci. Pharm. 76:567-598 (2008) for their description ofcyclodextrins and uses thereof in the pharmaceutical arts. As usedherein, “a cyclodextrin” or “cyclodextrins” refer not only tonaturally-occurring α-, β- and γ-cyclodextrins, but to cyclodextrinderivatives, including but not limited to those mentioned above.Likewise “α-cyclodextrin(s)”, “β-cyclodextrin(s)” and “γ-cyclodextrins”refer both to the naturally-occurring cyclodextrin and to cyclodextrinderivatives (e.g., “a β-cyclodextrin” includes both β-cyclodextrin andβ-cyclodextrin derivatives, such as, without limitation,hydroxypropyl-β-cyclodextrin, hydroxyethyl-β-cyclodextrin, randomlymethylated β-cyclodextrin, methyl-β-cyclodextrin,dimethyl-β-cyclodextrin, permethylated β-cyclodextrin, sulfobutyletherβ-cyclodextrin, branched β-cyclodextrin, etc.).

According to another non-limiting embodiment, the formulation is aliposome or multiphase (a liquid comprising more than one phase, such asoil in water, water in oil, liposomes or multi-lamellar structures)composition. Multi-phase systems, including liposomes, are prevalent inthe pharmaceutical arts. In the case of a liposome, the drug productmight comprise a phospholipid, a non-ionic detergent, and a cationiclipid, such as a composition comprising a phosphatidyl choline, anon-ionic surfactant, and a quaternary ammonium salt of alipid-substituted D or L glutamic acid or aspartic acid, and an aqueoussolvent. The liposomes or multiphase liquids and the ingredients thereofare pharmaceutically acceptable. They are typically formulated using anaqueous solvent, such as water, normal saline or PBS.

Phospholipids include any natural or synthetic diacylglycerylphospholiopid (such as phosphatidyl choline, phosphotidylethanolamine,phosphotidylserine, phosphatidylinositol, phosphatidylinositolphosphate, etc) and phosphosphingolipid that is capable of formingself-assembling liposomes. In one example the phospolipid is aphosphatidyl choline, a compound that comprises a choline head group,glycerophosphoric acid and fatty acid. Phosphatidyl choline can beobtained from eggs, soy or any suitable source and can be synthesized.

A nonionic surfactant is a surfactant containing no charged groups.Nonionic surfactants comprise a hydrophilic head group and a lipophilictail group, such as a single- or double-lipophilic chain surfactant.Examples of lipophilic tail groups include lipophilic saturated orunsaturated alkyl groups (fatty acid groups), steroidal groups, such ascholesteryl, and vitamin E (e.g., tocopheryl) groups, such as apolysorbate (a polyoxyethylene sorbitan), for example Tween 20, 40, 60or 80. More broadly, non-ionic surfactants include: glyceryl esters,including mono-, di- and tri-glycerides; fatty alcohols; and fatty acidesters of fatty alcohols or other alcohols, such as propylene glycol,polyethylene glycol, sorbitan, sucrose and cholesterol.

A cationic lipid is a compound having a cationic head and a lipophilictail. Included are cationic lipids that are quaternary ammonium salts,such as quaternary ammonium salts of lipid-substituted D and L glutamicacid or aspartic acid, such as glutamic acid dialkyl amides, includingfor example L-glutamic acid-1, 5,-dioleyl amide. Othercommercially-available examples of cationic lipids (e.g., available fromAvanti Polar Lipids) include DC-Cholesterol(313[N—(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride),DOTAP (e.g., 1,2-dioleoyl-3-trimethylammonium-propane (chloride salt)),DODAP (e.g., 1,2-dioleoyl-3-dimethylammonium-propane), DDAB (e.g.,Dimethyldioctadecylammonium (Bromide Salt)), ethyl-PC (e.g.,1,2-dilauroyl-sn-glycero-3-ethylphosphocholine (chloride salt)) andDOTMA (e.g., 1,2-di-O-octadecenyl-3-trimethylammonium propane (chloridesalt)).

The ratio of ingredients (phospholipid:nonionic surfactant:cationiclipid) can vary greatly, so long as a useful multilamellar structure isobtained that is able to deliver the active agents described herein.Further, each different combination of ingredients might have differentoptimal ratios. The ability to determine optimal ratios does not requireundue experimentation because the ability of any formulation to deliverthe active agent is readily tested as described herein, and as isgenerally known in the pharmaceutical arts. Liposome and multilamellarstructures are common delivery vehicles for active agents and theirmanufacture, physical testing and biological assays to determineeffectiveness are well-known. Useful phospholipid:nonionicsurfactant:cationic lipid ratios include, for example: from0.1-10:0.1-10:0.1-10 (w/w), and in certain instances the nonionicsurfactant:cationic lipid (w/w) ratio is approximately the same and/orthe phospholipid constituent is from 2 to 10 times (w/w) that of thenonionic surfactant and cationic lipid.

In use, a compound having the formula, e.g., as described herein:

in which

R is S, S(O), S(O)₂ or NH,

R1 is —NHR4 where R4 is OH, aminophenyl, hydroxyphenyl, C₁₋₄ alkylhydroxyphenyl or phenyl hydroxyphenyl, or —OR5 where R5 is H or C₁₋₄alkyl,R2 is phenyl, substituted phenyl,

where R5 is halo; methyl; C₁₋₄ alkyl; methoxy, C₁₋₄ alkoxy; naphthyl,1H-1,3-benzodiazol-2-yl, 1,3-benzothiazol-2-yl, pyrimidinyl,1-methyl-1H-1,3benzodiazol-2-yl, pyridyl, methoxyphenyl,methylthiophenyl, andR3 is from 0 to 5 methylene groups ((—CH₂-)₀₋₅) and 0 or 1 phenylenewherein at least one methylene or phenylene is present, or apharmaceutically acceptable salt thereof, whether or not incorporated asa drug product, is useful and finds use in: methods of improving kidneyfunction; methods of expanding renal progenitor cells in a kidney or invitro, for example a damaged kidney (including damaged, injured,defective or otherwise deficient); and methods of inhibiting a histonedeacetylase in a cell. In any such method, the compound or compositionis used in an amount effective to achieve the stated goal, whether it isimproving kidney function; expanding renal progenitor cells in a kidneyor in vitro or inhibiting a histone deacetylase in a cell. Suitableend-points for each method are provided herein. Where the method is forimproving kidney function in a patient, the method has an end point ofimproving one or more detectable parameters indicative of kidneyfunction, for example and without limitation moving abnormal Blood UreaNitrogen (BUN) levels in a patient towards normal levels, or expansionof renal progenitor cell populations in a kidney. Exemplary assaysshowing inhibition of histone acetylase are provided herein. Likewise,assays demonstrative of the ability of a compound to expand a renalprogenitor cell population are provided herein.

Example 1—Identification of PTBA as a Lead Compound

A chemical screen of approximately 2000 small molecules in zebrafishembryos identified a compound that generated pericardial edema,suggesting aberrant renal development. Treatment with this compound,4-(phenylthio)butanoic acid (PTBA), increased the size of the pronephrickidney in zebrafish. Earlier in development, PTBA expanded theexpression of renal progenitor cell markers, including lhx1a, pax2a, andpax8. Blocking DNA synthesis with hydroxyurea and aphidicolin beforePTBA treatment decreased its efficacy, suggesting that PTBA-mediatedrenal progenitor expansion is proliferation dependent.Structure-activity analysis revealed that PTBA was an analog of theknown histone deacetylase inhibitors (HDACis) 4-phenylbutanoic acid(PBA) and trichostatin A (TSA). Like PTBA, PBA and TSA both demonstratedthe ability to expand lhx1a expression in treated embryos. PTBA wassubsequently confirmed to function as an HDACi both in vitro and invivo. HDACis are hypothesized to stimulate retinoic acid (RA) signalingby decreasing the concentration of RA necessary to activate RA receptors(RARs) on target genes. Indeed, treatment with PTBA affected theexpression of the RA-responsive genes, cyp26a1 and cmlc2, in a mannerconsistent with increased RA signaling. Furthermore, blocking the RApathway with a dominant-negative RARα construct decreased PTBAefficiency. Therefore, PTBA appears to stimulate renal progenitor cellproliferation by activating the RA-signaling pathway. HDACis have beenshown to improve renal recovery following acute kidney injury. SincePTBA increases renal progenitor cell proliferation, it may exert similareffects on the multipotent cells involved in regeneration. In an effortto improve PTBA efficacy for pharmacological applications, analogs weregenerated by modifying the key structural elements of the general HDACipharmacophore. These were tested along with a panel of known HDACis fortheir ability to increase lhx1a expression in treated embryos. Severalcompounds were characterized that function at nanomolar concentrationsand do not cause toxicity in kidney cell culture. These secondgeneration PTBA analogs are excellent candidates for development aspotential renal therapeutics.

A scan was performed using an unbiased chemical library on zebrafishembryos, seeking small molecules capable of generating edemicphenotypes. Since edema may reflect renal dysfunction, it was hoped toidentify compounds capable of interfering with normal pronephricdevelopment (Drummond, I. A. Kidney development and disease in thezebrafish. J Am Soc Nephrol 16, 299-304, (2005)). Determining themechanisms of such compounds would provide insight into the molecularevents guiding the specification of renal progenitor cells. Of thealmost 2000 compounds tested, only four caused embryos to develop edemaby the 72 hours-post fertilization (hpf) endpoint. One of these,4-(phenylthio)butanoic acid (PTBA), demonstrated the ability to increasethe expression of renal progenitor cell markers. It is hypothesizedherein that PTBA increases the number of renal progenitor cells,resulting in aberrant pronephric development.

Results

PTBA Causes Edema in Zebrafish Larvae:

The initial chemical screen using a library of small molecules wasperformed with diverse structures. It was observed that 61 compounds(3%) were lethal and identified four compounds (NSC115787, NSC134664,NSC357777, and NSC35400, A-D in FIG. 2) that generated pericardial edemain treated zebrafish larvae at 72 hpf (FIG. 2, bottom panel).

In situ hybridization determined that treatment with NSC134664,NSC357777, or NSC35400 (a disconnected structure) did not affect thesize of the kidney field (data not shown). However, it was observed thattreatment with various concentrations of 4-(phenylthio) butanoic acid(PTBA, NSC115787) appeared to expand the expression of some renalmarkers during pronephric development (data not shown). PTBA wassynthesized and purified for use in all subsequent studies.

PTBA Expands the Kidney Field:

To determine the concentration of PTBA that maximized efficacy, whileminimizing toxicity, concentration-response experiments were performed.Treatment with 3 μM PTBA caused 92% (n=88) of the embryos to develop anedemic phenotype by 72 hpf without causing significant death (FIG. 3).Therefore, 3 μM PTBA was chosen as the working concentration. Allembryos treated with 10 μM PTBA (n=90), the concentration used in theinitial screen, died before 72 hpf (FIG. 3). This discrepancy probablyreflects the concentration variability common in small moleculelibraries.

The edemic phenotypes elicited by PTBA treatment prompted examination ofwhether the compound affects renal progenitor cells. To address this,expression patterns and relative abundance of lhx1a, pax2a, and pax8were determined at the 10-somite stage (14 hpf). This developmentalstage occurs just after specification of the first renal progenitorcells. Lhx1a expression was expanded in 95% of embryos treated with PTBA(n=60) as compared with controls (n=60) (FIGS. 4, A and B). Thisrepresented a three-fold increase in relative transcript as determinedby quantitative real-time PCR (qPCR; FIGS. 4, A and B). Increased lhx1aexpression appeared in the bilateral stripes of intermediate mesodermthat give rise to the pronephros (FIG. 4B, arrowheads), as well as inthe axial mesoderm (notochord; FIG. 4B, asterisk).

The expression domains of pax2a and pax8 were also expanded in 95% and97% of treated embryos, respectively, (n=60, pax2a; n=59, pax8) ascompared with controls (n=60, pax2a; n=59, pax8) (FIG. 4, C through F).This accounted for an almost two-fold increase in pax2a expression and a50% increase in pax8 expression as determined by qPCR (FIG. 4, C throughF). Although these studies demonstrated that PTBA treatment resulted inincreased gene expression, they did not indicate whether there are morerenal progenitor cells or simply higher expression levels per cell. Todifferentiate between these two possibilities, theTg(lhx1a:EGFP)^(pt303) reporter line (Swanhart, L. M. et al.Characterization of an lhx1a transgenic reporter in zebrafish. Int J DevBiol 54, 731-736, (2010)) was treated with PTBA and counted the numberof renal progenitor cells. As compared with control embryos,PTBA-treated embryos showed a 2.4-fold increase in the number of renalprogenitor cells (FIG. 5, A through H).

PTBA is Effective During Specification:

Initiation of PTBA treatments between 2 hpf and 14 hpf (10 somites)resulted in expanded pax2a expression at 24 hpf (FIG. 6, A through F).However, beginning treatment at 16.5 hpf (15 somites) resulted in nokidney field expansion (FIG. 6G). It was later determined thatinitiating treatments at 15 somites or later did not affect thefunctional kidney as assayed by a lack of edema in 72 hpf larvae (n=90,data not shown). Thus, the effective temporal treatment window exhibitedby PTBA coincides with the period when renal progenitor cells arespecified.

PTBA Increases Pronephric Size:

To determine whether PTBA treatment resulted in a transient orpersistent expansion of the kidney field, the kidney at 48 hpf wasexamined using markers of glomerulus and tubule. As compared withcontrols (n=54), 89% of PTBA-treated embryos (n=56) displayed anexpansion of the pan-tubule marker cdh17 (FIG. 7, A through D).Cross-sections from the proximal region of the cdh17 expression domainconfirmed the expansion (FIGS. 7, E and F). Pronephric expansion at theprotein level was also assessed by examining the expression ofNaK-ATPase, another pan-tubule marker (FIGS. 8, A and B). As comparedwith controls (n=10), 100% of PTBA-treated embryos (n=10) exhibitexpansion of NaK-ATPase protein expression. Cross-sections taken fromthe distal region of the NaK-ATPase expression domain show an increasein tubular diameter consistent with that observed with cdh17. (FIGS. 8,C and D, compare with FIGS. 7, E and F).

To determine if PTBA exhibits any segment-specific effects on the sizeof the pronephros, markers of podocytes (wt1a), proximal tubule(slc4a4), and distal tubule (slc12a1) were examined. As compared withcontrols (n=57, 58, and 56, respectively), PTBA-treated embryosexhibited 74% expansion of wt1a (n=50), 92% expansion of slc4a4 (n=60),and 64% expansion of slc12a1 (n=59) (FIG. 9). These results argue thatPTBA treatment likely causes expansion of the entire pronephros.Furthermore, while the wt1a expression domains in control embryos havemigrated to the midline by 48 hpf, the domains of PTBA-treated embryosremain separated (FIGS. 9, A and B). Since wt1a is essential for properglomerular morphogenesis, this misexpression may contribute to a loss ofkidney function. This observation could explain the edemic phenotypesassociated with PTBA treatment.

PTBA does not Transform Neighboring Tissues to a Kidney Fate:

The PTBA-mediated increase in kidney field size could result from thetransformation of nonrenal cells to a renal progenitor fate. To assessthis possibility, the effects of PTBA on markers of two mesodermaltissues juxtaposed to renal progenitor cells was examined: myod1(somites) and fli1a (vasculature). By in situ hybridization, it wasobserved that myod1 expression was slightly decreased in 95% ofPTBA-treated embryos (n=60) at the 10-somite stage, as compared withcontrols (n=60) (FIGS. 10, A and B). However, subsequent qPCR analysisdid not confirm the significance of this observed decrease (FIGS. 10, Aand B). Expression of fli1a remained unchanged in 97% of PTBA-treatedembryos (n=60), as compared with controls (n=59), and as assayed by qPCR(FIGS. 10, C and D).

In addition to increased lhx1a expression in renal progenitor cells,increased lhx1a expression in the notochord (FIG. 4B, asterisk) wasobserved. To determine whether this expansion reflected an effect onnotochord size or a general increase in lhx1a expression, thenotochord-specific marker ntla was assayed. It was observed that ntlawas increased in 88% of PTBA treated embryos (n=60), as compared withcontrols (n=59) (FIGS. 10, E and F). This represented an 80% increase inntla expression by qPCR analysis (FIGS. 10, E and F). The minimal effecton juxtaposed tissues coupled with an increase in the size of thenotochord suggests that these cell types are not being converted torenal progenitor cells. Therefore, PTBA treatment cannot be definitivelylinked to a fate-transformation event.

PTBA requires proliferation for efficacy: To examine the alternativepossibility that PTBA-mediated renal progenitor cell expansion dependson cell proliferation, the efficacy of PTBA was tested in the presenceof hydroxyurea and aphidicolin (HUA). HUA treatment has been previouslyshown to block cell division without affecting tissue specification(Harris, W. A. et al. Neuronal determination without cell division inXenopus embryos. Neuron 6, 499-515, (1991)). As expected, 97% ofPTBA-treated embryos (n=123) exhibited an expansion of lhx1a expressionat 10 somites, as compared with controls (n=136), (FIGS. 11, A and B).HUA treatment alone did not affect lhx1a expression (FIG. 11C). However,treatment with both HUA and PTBA resulted in lhx1a expansion in only 13%of 10-somite embryos (n=104) (FIG. 11D). Furthermore, although lhx1aexpression was decreased in the intermediate mesoderm, expression in theaxial mesoderm still appeared to be increased in treated embryos (FIG.11D). This result suggests that the PTBA-mediated lhx1a expansion in theaxial region is proliferation independent. Since lhx1a expression in theaxial mesoderm is gradually restricted to the tailbud duringsomitogenesis, the effect may reflect transcript perdurance.

Methods

Zebrafish Husbandry:

Zebrafish were maintained under standard conditions and staged aspreviously described (Kimmel, C. B., et al. Stages of embryonicdevelopment of the zebrafish. Dev Dyn 203, 253-310, (1995)). Embryoswere collected from group matings of wild-type AB adults. All animalhusbandry adhered to the National Institutes of Health Guide for theCare and Use of Laboratory Animals.

Small Molecule Screening:

The screen was performed in zebrafish embryos using the National CancerInstitute's Developmental Therapeutics Program (NCI/DTP) Diversity SetI. This library contains 1990 compounds selected by pharmacophoremodeling to represent the more than 140,000 small molecules maintainedin the NCI/DTP Open Repository. Compounds from the NCI/DTP Diversity SetI were diluted to 10 μM in E3 embryo medium (5 mM NaCl, 0.33 mM CaCl2,0.33 mM MgSO4, and 0.17 mM KCl) in a final DMSO concentration of 0.5%and arrayed in 96-well plates. Beginning at approximately 2 hpf, embryoswere transferred to each well in groups of five using a glass pipette.The plates were incubated at 28.5° C. for 70 hours. Individual wellswere then scored for a dominant phenotype, representative of at leastfour of the five embryos. The primary objective was to identifycompounds that caused edema in treated embryos at 72 hpf. Smallmolecules generating edema were retested once for verification, beforeobtaining additional compound from the NCI/DTP Open Repository.

Compound sources and treatments: PTBA was synthesized as describedbelow. Hydroxyurea and aphidicolin were obtained from Sigma-Aldrich.Groups of 20 to 30 chlorinated 2 hpf embryos were arrayed in individualwells of 12-well plates. E3 medium was removed with a glass pipette andreplaced with 1.5 ml treatment solutions containing 0.5% DMSO in E3 withor without compound at the reported concentrations. Treatments for allstudies were initiated at 2 hpf, except for the temporal studies (FIG.6) and the HUA studies (FIG. 11). HUA studies were performed asdescribed previously (Zhang, L., et al. Cell cycle progression isrequired for zebrafish somite morphogenesis but not segmentation clockfunction. Development 135, 2065-2070, (2008)), with the followingmodifications. HUA in 0.5% DMSO was added at early gastrulation (5 hpf)and PTBA was subsequently added at late gastrulation (8 hpf) to allowfor penetration of the proliferation inhibitors. All embryos wereincubated at 28.5° C. until the required developmental stage.

Synthesis of PTBA:

Methyl 4-(phenylthio)butanoate (1) was prepared from thiophenol,potassium carbonate, and methyl 4-bromobutyrate in refluxing acetone aspreviously described (Minoru, U., et al. Substituted thiobutyric acidderivatives (assigned to Otsuka Pharmaceutical Co., Ltd., Japan). Jpn.Kokai Tokkyo Koho (1982), 9 pp. JP 57-058663(A) Publication date1982-04-08 Showa, see FIG. 12). 4-(phenylthio)butanoic acid (2) wasprepared either in quantitative yield by saponification of 1 withaqueous KOH in MeOH overnight at room temperature followed byacidification with aqueous HCl, or in 97% yield from reaction of thesodium salt of thiophenol and γ-butyrolactone in refluxing EtOH andsubsequent acidification with aqueous HCl as previously described(Traynelis, V. J., et al. Seven-membered heterocycles. I. Synthesis ofbenzo[b]thiepin 1,1-dioxide and 1-phenylsulfonyl-4-phenyl-1,3-butadiene.J Org Chem 26, 2728-2733, (1961)). All compounds gave ¹H and ¹³C NMR(400/100 MHz and/or 600/150 MHz), mass spectra (GC-EI-MS, LC-ESI-MS, andhigh-resolution MALDI-TOF-MS), and melting points consistent with theliterature and their structures. All spectral and melting point datasuggested >99% purity.

Concentration-Response Studies:

Following 70 hours of treatment (as described above), edemic phenotypesin 72 hpf larvae were scored using a phenotype-based classificationsystem. Wild type: no visible edema or developmental delay. Edemic 1:pericardial edema evident, may exhibit slight developmental delay,little or no axis curvature, axis length normal. Edemic 2: pericardialedema evident, slight to moderate developmental delay, axis curvature,axis length normal or slightly reduced. Edemic 3: pericardial edemaevident, moderate to severe developmental delay, gross axis curvaturefrequently accompanied by tail kink, axis noticeably shortened.Significant effects (p<0.05) on survival were determined by two-tailedFisher's exact test in comparison with the 0 μM PTBA treatment group.

In situ hybridization and immunocytochemistry: In situ hybridization wasperformed as previously described with some modifications.142Hybridization temperature was 65° C. Embryos were blocked in 2% blockingreagent (Roche) with 5% sheep serum in MAB (100 mM maleic acid and 150mM NaCl [pH 7.5]). Whole-mount immunocytochemistry with 1:25 mouseanti-α6F antibody (Developmental Studies Hybridoma Bank) and 1:100 Cy3secondary antibody (Jackson ImmunoResearch) was performed as describedpreviously.108 Embryos were embedded in JB-4 for sectioning per themanufacturer's instructions (Polysciences), sectioned at 5 μm, andmounted with Cytoseal 60 (Richard-Allan Scientific).

Relative qPCR.

Several of the genes analyzed by qPCR, including lhx1a and pax2a, showexpression in anterior regions of the embryo as well as the IM. To focusmore specifically on effects in the IM, cDNA samples taken from thetrunks of treated and control embryos were examined. Samples for trunkRNA extraction were prepared by cutting embryos just above the firstsomite with microscissors and discarding the anterior portion. The trunkportions were homogenized with a plastic microcentrifuge pestle in 500μL of TRI reagent (Ambion), and RNA was isolated using an RNeasy MicroKit (QIAGEN) per the manufacturer's instructions. 1 μg RNA was heated to75° C. for 5 min and then placed on ice. The following reagents werethen added to a final volume of 29 μI: 1× Expand High-Fidelity PCRbuffer without MgCl2 (Roche), 3 mM MgCl2, 500 μM dNTPs, 3.3 μM randomhexamers, and 30 U Protector RNase Inhibitor (Roche). The mixture waspreincubated to 42° C. for 5 min. 1 μl of 200 U/μ1 SuperScript IIReverse Transcriptase (RT) or RNase-free water was added for +RT or −RTreactions, respectively. Reactions were incubated at 42° C. for 1 hr andthen stopped by heating to 95° C. for 5 min. Reaction products werestored at −20° C.

Primer sets were designed using NetPrimer and Beacon Designer (ver.7.51) primer analysis software (PREMIER Biosoft). In each set, oneprimer was designed to span an exon boundary. In addition, at least oneprimer was confirmed to exhibit no significant cross-homology whencompared against the NCBI zebrafish RefSeq mRNA library by BLAST search.Primer melting temperatures were maintained between 60° C. and 64° C. asdetermined by NetPrimer. Each primer set was observed to generate asingle amplicon of expected length following qPCR. The reference geneprimer sets have been previously described (Tang, R., Dodd, A., Lai, D.,McNabb, W. C. & Love, D. R. Validation of zebrafish (Danio rerio)reference genes for quantitative real-time RT-PCR normalization. ActaBiochim Biophys Sin (Shanghai) 39, 384-390, (2007)) with onemodification. The β-actin (F) sequence was changed to:CGTGCTGTCTTCCCATCCA (SEQ ID NO: 1). This corrects a one base discrepancyfrom the reported ENSDART accession number. The other primer setsincluded:

lhx1a (F): (SEQ ID NO: 2) TTCATACTATGGAGATTATCAAAGCG, lhx1a (R):(SEQ ID NO: 3) GGTCCTGATGAGGGAACAAAAG, pax2a (F): (SEQ ID NO: 4)GTCCCTGGAAGCGACTTTTC, pax2a (R): (SEQ ID NO: 5) TTGACTGGGCTGCGATGG,pax8 (F): (SEQ ID NO: 6) GCTCCGCCGTCACTCCTC, pax8 (R): (SEQ ID NO: 7)TCTCCTGGTCACTGTCATCGTG, ntla (F): (SEQ ID NO: 8)CGCAGCACTACCACCAATAACTAC, ntla (R): (SEQ ID NO: 9)GAGCCTGATGGGGTGAGAGTC, myod1 (F): (SEQ ID NO: 10)TTCTGGAACATTACAGTGGAGACTC, myod1 (R): (SEQ ID NO: 11)GTGCGTCAGCATTTGGTGTG, fli1a (F): (SEQ ID NO: 12) CGGAAAAGGCTCTCCAACAG,fli1a (R): (SEQ ID NO: 13) TGCTGGTGGGTCCTAATATCTG.

Relative qPCR was performed as described previously (Tang, R., et al.Acta Biochim Biophys Sin (Shanghai) 39, 384-390, (2007)) with somemodifications. 25 μl reactions were prepared containing the followingreagents: 12.5 μl 2×iQ SYBR Green Supermix (Bio-Rad), 5 μl 1 μM primermix (1 μM each of forward and reverse primer), 5.5 μl RNase-free water,and 2 μl 1:10-diluted template (+RT or −RT product) or 2 μl RNase-freewater (no template control). Each assay was performed in triplicatewells using an iQ5 Real-Time PCR Detection System (Bio-Rad). Thermalcycling was performed for 40 cycles, each consisting of 94° C. for 15 s,then 59° C. for 1 min. Following amplification, melt curve analysis wasperformed to assess non-specific amplification. Each primer set yieldeda single peak, indicative of specific amplification. Reactions performedusing −RT product or no template controls were observed to exhibitlittle or no amplification in comparison with their +RT counterparts.

Seven reference gene candidates [β-actin, β2 microglobulin, elongationfactor 1 alpha, hypoxanthine guanine phosphoribosyl transferase 1, RNApolymerase subunit D, ribosomal protein L13a, and succinatedehydrogenase complex subunit A (SDHA)] (Tang, R., et al. Validation ofzebrafish (Danio rerio) reference genes for quantitative real-timeRT-PCR normalization. Acta Biochim Biophys Sin (Shanghai) 39, 384-390,(2007)) were screened to determine the gene(s) least affected by PTBAtreatment. Relative qPCR experiments (n=3, 180 embryos) were performedusing trunk cDNA obtained from 10-somite embryos treated from 2 hpf witheither 0.5% DMSO or 3 μM PTBA. The results were analyzed usingNormFinder software (ver. 0.953) to determine the most stable referencegene or combination of genes (Andersen, C. L., Jensen, J. L. & Orntoft,T. F. Normalization of real-time quantitative reverse transcription-PCRdata: a model-based variance estimation approach to identify genessuited for normalization, applied to bladder and colon cancer data sets.Cancer Res 64, 5245-5250, (2004)). The combination of β-actin and SDHAwas observed to exhibit the most stability, and was therefore used fornormalization of all qPCR data.

Relative gene expression was calculated using iQ5 software (ver. 2.0,Bio-Rad) to determine normalized expression levels (ΔΔCt method). Forcomparison of fold-differences, the expression levels obtained fromDMSO-treated controls were set to a value of 1.0. The amplificationefficiency of each reaction was calculated using LinRegPCR software(ver. 11.4, Ruijter, J. M. et al. Amplification efficiency: linkingbaseline and bias in the analysis of quantitative PCR data. NucleicAcids Res 37, e45, (2009)). The mean efficiencies of each tested primerset fell between 91% and 100%. Mean expression levels (normalized to thecontrol group) and the corrected expression SD were used to generate 95%confidence intervals for each data set.

Cell Counting:

Tg(lhx1a:EGFP)^(pt303) embryos were treated with 3 μM PTBA and thenfixed in 4% paraformaldehyde in PBS for 8 hours at 4° C. Embryos werewashed in PBS containing 0.1% TWEEN 20 (PBT) and incubated in 1 μg/mlDAPI in PBT for 30 minutes at room temperature. Embryos wereflat-mounted on glass slides with Cytoseal 60 and imaged with either aLeica M205 FA epifluorescent microscope or an Olympus FluoView 1000confocal microscope. Confocal projections contained stacks of six 3 μmimages. A predefined box was positioned at the most posterior region ofthe notochord and included the most lateral GFP-positive cell in akidney field. The cells that were positive for both GFP and DAPI withinthis box were counted manually with the aid of ImageJ. Variances of thecontrol and PTBA-treated groups were compared by F test and found to beunequal. Therefore, a two-tailed t test with unequal variance was usedto determine significance (α=0.05).

Example 2—Mechanism of TBA Efficacy

Having established that PTBA stimulates renal progenitor cellproliferation, the mechanism responsible for this effect was thendetermined. Initial structure-activity relationship experimentsdemonstrated that certain structural motifs within PTBA modify itsefficacy. While these results provided important leads for future analogdevelopment, they did not reveal any clues to the underlying mechanism.The breakthrough came when subsequent analysis revealed that PTBA isstructurally similar to known histone deacetylase inhibitors (HDACis),including 4-phenylbutanoic acid (PBA) and trichostatin A (TSA). HDACisare thought to attenuate retinoic acid receptor-mediated inhibition oftarget genes, lowering the threshold of retinoic acid (RA) required toactivate transcription. Since RA affects pronephric development, it washypothesized that PTBA functions as an HDACi and its effects aremediated through the RA pathway.

Results

PTBA Structure-Activity Studies Reveal Critical Motifs:

Structure-activity analyses was performed using a series of sevenanalogs (FIG. 13). In situ hybridization for lhx1a was performed on10-somite embryos treated with each analog at 3 μM. The results werecompared with control (no expansion, n=53) (FIG. 13A) and PTBA-treatedembryos (100% expansion, n=52) (FIG. 13B). Replacement of the phenylthioether with a phenylsulfonyl linkage stripped the compound of its effectson renal progenitor cells (no expansion, n=54) (FIG. 13C). Therefore,the oxidation state of the sulfur atom is an activity determinant.However, 4-(naphthalen-2-yl thio)butanoic acid, an analog carrying anaphthalene ring in place of the phenyl moiety of PTBA, still expandslhx1a expression to some extent (33% expansion, n=39) (FIG. 13D). Thus,modifications of the ring structure are tolerated and suggest a site forfuture analog development. Two analogs containing substitutions of thebutanoic acid backbone, 2-amino-PTBA and 3-(phenylthio)benzoic acid hadno effect on lhx1a expression (no expansion, n=64 and n=53,respectively) (FIGS. 13, E and F), suggesting a requirement for aflexible hydrocarbon backbone for biological activity. 4-phenoxybutanoicacid and 5-phenylpentanoic acid, which contain oxygen and carbonsubstitutions for the sulfur atom, respectively were examined.4-Phenoxybutanoic acid exhibited reduced activity compared with theparent compound (13% expansion, n=56) (FIG. 13G), while5-phenylpentanoic acid was inactive (no expansion, n=55) (FIG. 13H).These results suggest that compound efficacy is improved when an atomwith a nonbonding electron pair(s) occupies this position. Finally, theesterified analog methyl-4-(phenylthio)butanoate exhibited equal potencyto PTBA (100% expansion, n=41) (FIG. 13I).

HDACis Mimic the Effects of PTBA:

Subsequent structural analysis revealed that PTBA is a close analog of4-phenylbutanoic acid (PBA), a known HDACi (FIGS. 14, A and B), and thatboth compounds exhibit some similarity to the HDACi trichostatin A (TSA)(FIG. 14C). Indeed, all three structures contain the elements of theHDACi pharmacophore, a general representation of the functional domainswithin this class of compounds (FIG. 14D). These include an aliphatic oraromatic cap, connecting unit, hydrophobic linker, and zinc-bindinggroup.

Because PBA and TSA are structurally analogous to PTBA, it wasdetermined whether they shared the same ability to expand renalprogenitor cells. Concentration-response experiments were performed todetermine the concentrations of PBA or TSA necessary to elicit lhx1aexpansion, if any (FIGS. 15 and 16).

It was determined that treatment with 25 μM PBA or 200 nM TSA producedan expansion of lhx1a expression consistent with that elicited by 3 μMPTBA (FIGS. 15E and 16D compared with FIG. 4B). Therefore, at least twoknown HDACis mimic the effects of PTBA, supporting the idea that PTBAlikewise functions as an HDACi.

PBA and TSA Exhibit Greater Toxicity than PTBA:

How trunk mesoderm juxtaposed to the kidney field is affected by PBA andTSA treatments was then examined. PTBA treatment does not significantlyaffect the expression of somitic or vascular markers (FIG. 10, A throughD) Furthermore, although PTBA increased ntla expression, the generalstructure of the notochord remained relatively unchanged (FIGS. 10, Eand F). However, TSA is a broad-spectrum HDACi that causes disruption ofmultiple tissues in zebrafish and demonstrates renal toxicity in cellculture. Therefore, embryos were treated with 25 μM PBA or 200 nM TSAand the expression of myod1, fli1a, and ntla was compared with that ofcontrol embryos. Expression of myod1 was decreased in 57% of PBA-treatedembryos (n=53) and 100% of TSA-treated embryos, as compared to controls(n=54) (FIG. 17, A through C). Furthermore, TSA treatment caused analmost complete loss of the somitic blocks (FIG. 17C). As compared withcontrols (n=54), fli1a expression decreased in 78% of PBA-treatedembryos (n=54) and 95% of TSA-treated embryos (n=55) (FIG. 17, D throughF). PBA increased ntla expression in 87% of treated embryos (n=52), ascompared to controls (n=55) (FIGS. 17, G and H). This effect appearssimilar to the expanded ntla expression observed following PTBAtreatment (FIG. 10F). In contrast, TSA disrupted normal ntla expressionin 86% of treated embryos (n=56), as compared with controls (FIGS. 17, Gand I). This resulted in breaks in the ntla expression pattern (FIG.17I, arrowheads).

To determine the toxicity of PBA and TSA relative to PTBA, phenotypicconcentration-response experiments were again performed. 72 hpf larvaewere assessed for the development of edemic phenotypes over the sameconcentration range used to test for expansion of the kidney field.Pericardial edema was evident in 23% of larvae treated with 25 μM PBA(n=83) and 100% of larvae treated with 200 nM TSA (n=35) (FIGS. 18 and19). In addition, 25 μM PBA caused minimal, but significant death (8%,n=90), while treatment with 200 nM TSA resulted in high lethality (61%,n=90) (FIGS. 18 and 19).

PTBA Functions as an HDACi In Vitro:

Because PBA and TSA mimic the ability of PTBA to expand renal progenitorcells, it was determined whether PTBA functions as an HDACi. To evaluatethis in vitro, the deacetylation of a fluorescent peptide substrate wasmeasured in the presence of human HDACs. HDAC activity increased indirect proportion to the amount of HeLa cell nuclear extract added tothe assay (FIG. 20, black triangle). Addition of TSA completely blockedactivity at all input levels of nuclear extract (FIG. 20, graytriangle). Previous work showed that 5 mM PBA decreased HDAC activity inDSI9 mouse erythroleukemia cells to 19% of the control value (Lea, M. A.& Tulsyan, N. Discordant effects of butyrate analogues onerythroleukemia cell proliferation, differentiation and histonedeacetylase. Anticancer Res 15, 879-883, (1995)). To determine whetherPTBA inhibited HDACs to a similar extent as PBA, both compounds wereevaluated at 5 mM. PTBA and PBA showed similar potency, reducing theHDAC activity elicited by 10 μg HeLa extract to 30% of the control value(FIG. 20, diamond and circle, respectively). Previously, the PTBAanalog, 4-(phenylsulfonyl)butanoic acid (PSOBA), demonstrated no abilityto expand renal progenitor cells (FIG. 13C). Therefore, it washypothesized that it would function poorly in vitro as an HDACi. Asexpected, 5 mM PSOBA decreased the HDAC activity elicited by 10 μg HeLaextract to only 70% of the control value (FIG. 20, square).

PTBA Functions as an HDACi In Vivo:

It was next determined whether the HDACi function of PTBA wasquantifiable in vivo. 24 hpf embryos were treated with 3 μM PTBA, 25 μMPBA, or 200 nM TSA for 6 hours. Protein extracts were prepared andimmunoblotted with an anti-hyperacetylated histone H4 antibody. HistoneH4 hyperacetylation was observed following treatment with all threecompounds at their tested concentrations, as compared with the control(FIG. 21). Furthermore, increasing the compound concentration caused acorresponding increase in hyperacetylation (FIG. 21). Therefore, both invitro and in vivo results confirm that PTBA functions as an HDACi.

PTBA Affects Retinoic Acid Signaling:

HDACis are believed to lower the threshold of RA necessary to activatetranscription. If PTBA treatment facilitates activation of the RApathway, then expression of genes responsive to RA signaling shouldchange. Consequently, two genes were focused on: cyp26a1, which isdirectly activated by RA signaling, and the cardiac gene cmlc2, whoseexpression in the heart field size is reduced by RA treatments. In aprevious study, cmlc2 expression was assessed for RAdependent effects in18-somite embryos (Keegan, B. R., Feldman, J. L., Begemann, G., Ingham,P. W. & Yelon, D. Retinoic acid signaling restricts the cardiacprogenitor pool. Science 307, 247-249, (2005)), therefore embryos werecollected at the 18-somite stage for continuity. As compared to controls(n=58), expression of cyp26a1 was increased in 100% of PTBA-treatedembryos (n=57) (FIGS. 22, A and B). In agreement with this result, cmlc2expression was decreased in 100% of PTBA-treated embryos (n=58), ascompared to controls (n=57) (FIGS. 22, C and D).

To provide a stronger link between PTBA treatment and RA signaling, mRNAencoding a dominant-negative RARα construct (DN-RARα), which is known toblock RA signaling (Blumberg, B. et al. An essential role for retinoidsignaling in anteroposterior neural patterning. Development 124,373-379, (1997)), was injected prior to PTBA treatment. In situhybridization was performed to determine the maximum amount of DN-RARαmRNA that could be injected at the one-cell stage without affectinglhx1a expression. In the tested range of 0 to 400 ng, embryos injectedwith 200 ng DNRARα mRNA or less showed normal lhx1a expression, whilehigher doses caused aberrant or decreased expression (data not shown).Therefore, 200 ng DN-RARα mRNA was injected to assess the relationshipbetween PTBA and the RA pathway. As compared with controls (n=80), anexpansion of lhx1a expression was observed in 90% of the mock-injectedPTBA-treated embryos at the 10-somite stage (n=78) (FIGS. 23, A and B).Expression of lhx1a appeared normal in 92% of embryos injected with 200pg DN-RARα mRNA (n=125) (FIG. 23C). However, only 19% of the embryosinjected with 200 pg DN-RARα mRNA and subsequently treated with PTBAshowed expanded lhx1a expression (n=125) (FIG. 23D). Therefore, thesedata suggest that PTBA-mediated expansion of renal progenitor cells isdependent on the retinoic acid pathway.

Methods

Zebrafish husbandry and in situ hybridization were performed asdescribed above.

Compound Sources and Treatments:

PTBA and methyl-4-(phenylthio)butanoate were synthesized as describedabove. 4-(Naphthalen-2-ylthio)butanoic acid (NSC2733),3-(phenylthio)benzoic acid (NSC113994), and 2-amino-PTBA (NSC140113)were obtained from the NCI/DTP Open Repository. PBA, TSA,4-phenoxybutanoic acid, and 5-phenylpentanoic acid were obtained fromSigma-Aldrich. PSOBA was obtained from Matrix Scientific. Treatmentswere performed as described above, except for the in vivohyperacetylation assays and DN-RARα experiments (see below).

Histone Hyperacetylation Assays:

SDS-PAGE and Western blotting were performed as described previouslywith some modifications (Noel, E. S. et al. Organ-specific requirementsfor Hdac1 in liver and pancreas formation. Dev Biol 322, 237-250,(2008)). Proteins were separated on 18% SDS-PAGE gels. Membranes wereincubated at 4° C. overnight with 1:1000 anti-hyperacetylated histone H4antibody (06-946, Millipore) or 1:1000 anti-α-tubulin antibody(Sigma-Aldrich) in PBT containing 5% nonfat milk.

Fluorescence HDAC Assays:

In vitro HDAC activity assays were performed using a fluorescence HDACassay kit (Active Motif) according to the manufacturer's instructions.For maintaining compound solubility at 5 mM, the final DMSOconcentration in all assay wells was increased to 5%. Fluorescence wasdetected using an M5 Plate Reader (Molecular Dynamics)

mRNA Synthesis and Microinjections:

Synthetic mRNA was generated from the XRARα1⁴⁰⁵/pCD61 construct(Blumberg, B. et al. An essential role for retinoid signaling inanteroposterior neural patterning. Development 124, 373-379, (1997),NotI digested) using a T7 mMessage mMachine kit (Ambion). Zebrafishembryos were injected at the one-cell stage either with 200 pg ofsynthetic mRNA and 1% fluorescein dextran (Sigma-Aldrich) or with 1%fluorescein dextran alone (mock) and allowed to develop in E3 culturemedium at 28.5° C. At the 256-cell stage, only fluorescein-dextranpositive embryos were selected for PTBA treatment, which occurred at 5hpf.

Example 3—Development of PTBA Analogs

Determining that treating zebrafish embryos with PTBA, a novel HDACi,increased the number of renal progenitor cells leading to acorresponding increase in pronephric size suggested that these cells arecapable of contributing to nephrogenesis. As early as 1989, Bacallo andFine proposed that kidney regeneration follows the same pattern ofdifferentiation events that lead to nephrogenesis (Molecular events inthe organization of renal tubular epithelium: from nephrogenesis toregeneration. Am J Physiol 257, F913-924, (1989)). Both processes beginwith the proliferation of renal progenitor cells to provide the rawmaterial necessary for subsequent differentiation into kidney tissue.Since PTBA stimulates renal progenitor cell proliferation duringpronephric development, it may function similarly during kidneyregeneration. However, developing PTBA into a potential therapeuticrequires the consideration of both its efficacy and toxicity.Furthermore, expanding the structure-activity relationship study to awider selection of small molecules may yield better candidates forfuture in vivo studies. Therefore, a panel of structural and functionalanalogs of PTBA was evaluated to identify compounds exhibiting nanomolarefficacy and low toxicity in kidney cell culture. It was hypothesizedthat modifying the key structural elements that determine the HDACiactivity of PTBA would improve its ability to expand renal progenitorcells.

Results

Phenotypic Screening of PTBA Analogs:

As shown above, treating zebrafish embryos with 3 μM PTBA generatespericardial edema by 72 hpf (FIG. 3). This PTBA concentration alsostimulates the proliferation of renal progenitor cells during pronephricdevelopment (FIGS. 5 and 11). Furthermore, by 48 hpf, embryos treatedwith 3 μM PTBA exhibit wider pronephric tubules and a failure of wt1aconvergence at the dorsal midline (FIGS. 7, 8, and 9) Therefore, it isreasonable to hypothesize that the edemic phenotype reflects aberrantkidney morphogenesis resulting from an overabundance of renal progenitorcells. Consequently, a phenotypic screen was performed on a panel ofPTBA analogs at 3 μM to identify potentially effective compounds. Eachof the compounds chosen for analysis represents either a structural orfunctional analog of the lead compound, PTBA. The structural analogscontain functional group additions or substitutions in one of fourelements of the PTBA structure. These modify the key determinants ofHDACi activity as predicted by the general pharmacophore: the aliphaticcap, connecting unit, hydrophobic linker, and zinc-binding group (FIGS.14D and 24). The structural analogs selected for the panel each containone or more of these modifications (Table 2). Several functional groupchoices were based on previous structure-activity relationship studiesof PTBA. For example, methylating the zinc-binding group of PTBAgenerated a compound equally capable of expanding lhx1a expression intreated embryos (FIG. 13I). Therefore, the efficacy of several alkylatedanalogs was assessed.

TABLE 2 Structural analogs of PTBA (See, FIG. 24). Aliphatic ConnectingFatty Acid Zinc-Binding Compound Name Cap (W) Unit (X) Linker (Y) Group(Z) 4-(phenylthio)butanoic acid (PTBA) H S CH₂ OH4-(naphthalen-2-ylthio)butanoic acid C₆H₆ S CH₂ OH4-(phenylsulfonyl)butanoic acid H SO₂ CH₂ OH 4-phenoxybutanoic acid H OCH₂ OH 4-(phenylamino)butanoic acid H NH CH₂ OH 5-phenylpentanoic acid HCH₂ CH₂ OH 3-(phenylthio)benzoic acid H S C₆H₆ OH methyl4-(phenylthio)butanoate H S CH₂ OCH₃ propyl 4-(phenylthio)butanoate H SCH₂ O(CH₂)₂CH₃ butan-2-yl 4-(phenylthio)butanoate H S CH₂ OCH(CH₃)CH₂CH₃tert-butyl 4-(phenylthio)butanoate H S CH₂ OC(CH₃)₃N-hydroxy-4-(phenylthio)butanamide H S CH₂ NHOH methyl4-[(4-methylphenyl)thio]butanoate CH₃ S CH₂ OCH₃ methyl4-[(4-methoxyphenyl)thio]butanoate OCH₃ S CH₂ OCH₃ methyl4-[(4-fluorophenyl)thio]butanoate F S CH₂ OCH₃ methyl4-[(4-chlorophenyl)thio]butanoate Cl S CH₂ OCH₃ methyl4-[(4-bromophenyl)thio]butanoate Br S CH₂ OCH₃N-hydroxy-4-[(4-methylphenyl)thio]butanamide CH₃ S CH₂ NHOHN-hydroxy-4-[(4-methoxyphenyl)thio]butanamide OCH₃ S CH₂ NHOH4-[(4-fluorophenyl)thio]-N-hydroxybutanamide F S CH₂ NHOH4-[(4-chlorophenyl)thio]-N-hydroxybutanamide Cl S CH₂ NHOH4-[(4-bromophenyl)thio]-N-hydroxybutanamide Br S CH₂ NHOH

In addition, it was observed that the hydroxamic acid HDACi,trichostatin A (TSA), exhibited high efficacy, increasing lhx1aexpression at 200 nM. (FIG. 16D). Hydroxamic acids, which form twocoordinate bonds with the active site zinc, are generally stronger thancarboxylic acids, including PTBA, which form only one (Jacobsen, F. E.,Lewis, J. A. & Cohen, S. M. The design of inhibitors for medicinallyrelevant metalloproteins. Chem Med Chem 2, 152-171, (2007)). Thus,several of the analogs tested contained hydroxamate moieties on thezinc-binding group. Ten functional analogs were also chosen to representa subset of known HDACis of several different classes (FIG. 25). Theseinclude inhibitors derived from carboxylic acids, hydroxamic acids,benzamides, and natural products (Villar-Garea, A. & Esteller, M.Histone deacetylase inhibitors: understanding a new wave of anticanceragents. Int J Cancer 112, 171-178, (2004)). With the exception ofbutanoic acid, all of the chosen compounds have predicted octanol-waterpartition coefficients (Log Ps) greater than 1 (Table 3).

TABLE 3 Predicted octanol-water partition coefficients (XLogPs) of thePTBA analogs. XLogP values were calculated from the chemical structuresusing XLOGP3 (ver. 3.2.2), a web-based application. Compound Name XLogPbutanoic acid 0.79 4-(phenylsulfonyl)butanoic acid 1.014-(phenylamino)butanoic acid 1.41N-hydroxy-4-[(4-methoxyphenyl)thio]butanamide 1.52 APHA compound 8 1.52N-hydroxy-4-(phenylthio)butanamide 1.554-[(4-fluorophenyl)thio]-N-hydroxybutanamide 1.65 SAHA 1.86N-hydroxy-4-[(4-methylphenyl)thio]butanamide 1.91 MS-275 2.024-phenoxybutanoic acid 2.14 4-[(4-chlorophenyl)thio]-N-hydroxybutanamide2.18 Scriptaid 2.18 4-[(4-bromophenyl)thio]-N-hydroxybutanamide 2.244-(phenylthio)butanoic acid (PTBA) 2.29 4-phenylbutanoic acid (PBA) 2.42methyl 4-[(4-methoxyphenyl)thio]butanoate 2.59 methyl4-(phenylthio)butanoate 2.61 S-phenylpentanoic acid 2.70 methyl4-[(4-fluorophenyl)thio]butanoate 2.71 valproic acid 2.75 trichostatin A(TSA) 2.75 methyl 4-[(4-methylphenyl)thio]butanoate 2.98 methyl4-[(4-chlorophenyl)thio]butanoate 3.24 methyl4-[(4-bromophenyl)thio]butanoate 3.31 propyl 4-(phenylthio)butanoate3.51 4-(naphthalen-2-ylthio)butanoic acid 3.54 tert-butyl4-(phenylthio)butanoate 3.60 butan-2-yl 4-(phenylthio)butanoate 3.94apicidin 4.41 3-(phenylthio)benzoic acid 4.45 tubacin 6.34

Log P indicates the hydrophobicity of a given molecule, with lipophiliccompounds exhibiting higher values (Lipinski, C. A., et al. Experimentaland computational approaches to estimate solubility and permeability indrug discovery and development settings. Adv Drug Deliv Rev 46, 3-26,(2001) and Testa, B., et al. Lipophilicity in molecular modeling. PharmRes 13, 335-343, (1996)). Previous work determined that zebrafishembryos absorb compounds with log P values between 1 and 12 from embryomedium. The 32 compounds and a DMSO control were examined for theirability to generate pericardial edema in zebrafish larvae by 72 hpf(Table 2). Treatment with 12 compounds did not cause astatistically-significant decrease in the number of wild-type larvalphenotypes, as compared with controls. This group included the fourcompounds containing connecting unit substitutions, seven of the knownHDACis, and one alkylated analog, tert-butyl 4-(phenylthio)butanoate.Treatment with each of the remaining analogs generated edemic and/orlethal phenotypes that were scored using the phenotype-basedclassification system described above. Three of these compounds, TSA,valproic acid, and apicidin, are known HDAC inhibitors. The lethality of3 μM TSA was expected, since it was previously demonstrated that 300 nMTSA killed greater than 90% of treated 72 hpf larvae (FIG. 19).

TABLE 4 Phenotypes observed in larvae treated with PTBA analogs. Embryoswere treated with each compound at 3 μM from 2 hpf, and larvae werescored at 72 hpf using a phenotype-based classification system describedabove). Compounds below the indicated line exhibit a significantdecrease (p < 0.05) in the occurrence of wild-type phenotypes asdetermined by Fisher's exact test. Wild Type Edemic 1 Edemic 2 Edemic 3Compound Name (WT) (E1) (E2) (E3) Dead 0.5% DMSO 36 0 0 0 04-phenoxybutanoic acid 36 0 0 0 0 4-(phenylsulfonyl)butanoic acid 36 0 00 0 5-phenylpentanoic acid 36 0 0 0 0 APHA compound 8 36 0 0 0 0 MS-27536 0 0 0 0 PBA 36 0 0 0 0 SAHA 36 0 0 0 0 tubacin 36 0 0 0 04-(phenylamino)butanoic acid 35 1 0 0 0 Scriptaid 35 1 0 0 0 butanoicacid 35 0 0 0 1 tert-butyl 4-(phenylthio)butanoate 33 3 0 0 0 Thecompounds below this line cause significant decrease in wild-type larvalphenotypes at 3 μM (p < 0.05) valproic acid 25 11 0 0 04-(naphthalen-2-ylthio)butanoic acid 25 9 0 0 2N-hydroxy-4-[(4-methoxyphenyl)thio]butanamide 16 20 0 0 0 methyl4-[(4-bromophenyl)thio]butanoate 8 27 1 0 0 3-(phenylthio)benzoic acid 234 0 0 0 4-[(4-bromophenyl)thio]-N-hydroxybutanamide 1 23 12 0 0butan-2-yl 4-(phenylthio)butanoate 2 19 12 2 1 PTBA 0 13 17 5 1 methyl4-[(4-chlorophenyl)thio]butanoate 0 10 6 19 1 methyl4-[(4-methoxyphenyl)thio]butanoate 0 2 24 9 1N-hydroxy-4-(phenylthio)butanamide 0 3 20 13 0 propyl4-(phenylthio)butanoate 0 7 8 15 6 methyl 4-(phenylthio)butanoate 0 0 226 8 Methyl 4-[(4-methylphenyl)thio]butanoate 0 0 2 23 114-[(4-chlorophenyl)thio]-N-hydroxybutanamide 0 0 0 22 14N-hydroxy-4-[(4-methylphenyl)thio]butanamide 0 0 0 14 224-[(4-fluorophenyl)thio]-N-hydroxybutanamide 0 0 0 8 28 methyl4-[(4-fluorophenyl)thio]butanoate 0 0 0 0 36 apicidin 0 0 0 0 36 TSA 0 00 0 36

Furthermore, with the exception of apicidin, all of the compounds thatcause more severe phenotypes than PTBA contain either hydroxamic oralkylated zinc-binding groups. Taken together, these results suggestthat modifications of PTBA anticipated to alter its underlying HDACifunction may affect compound efficacy during pronephric development.However, pericardial edema can develop as a result of organ dysfunctionunrelated to the kidney. Therefore, the effect of each of thesecompounds, if any, on renal progenitor cells was determined. Toaccomplish this, lhx1a expression was examined in treated embryos at the10-somite stage. In situ hybridizations was performed on embryos treatedwith decreasing concentrations of compound, beginning at 3 μM (Table 5).The results were categorized relative to the efficacy of PTBA at a givenconcentration and are detailed below.

TABLE 5 Lhx1a expansion caused by analog treatment. In situhybridization for lhx1a expression in 10-somite embryos treated from 2hpf with each compound at the listed concentration. Analogs wereclassified according to their ability to increase lhx1a expression aseffective (+), partially effective (+/−), or ineffective (−). Analogsthat killed all embryos at a given concentration before reaching 10somites are listed as XX. If the efficacy of a compound was notdetermined at a given concentration it is listed as ND. Compound Name 3μM 1.5 μM 800 nM 400 nM 200 nM 100 nM 0.5% DMSO − − − − − −4-phenoxybutanoic acid − ND ND ND ND ND 4-(phenylsulfonyl)butanoic acid− ND ND ND ND ND 5-phenylpentanoic acid − ND ND ND ND ND APHA compound 8− ND ND ND ND ND MS-275 − ND ND ND ND ND PBA − ND ND ND ND ND SAHA − NDND ND ND ND tubacin − ND ND ND ND ND 4-(phenylamino)butanoic acid − NDND ND ND ND Scriptaid +/− ND ND ND ND ND butanoic acid − ND ND ND ND NDtert-butyl 4-(phenylthio)butanoate +/− ND ND ND ND ND valproic acid +/−ND ND ND ND ND 4-(naphthalen-2-ylthio)butanoic acid − ND ND ND ND NDN-hydroxy-4-[(4- +/− ND ND ND ND ND methoxyphenyl)thio]butanamide methyl4-[(4- +/− ND ND ND ND ND bromophenyl)thio]butanoate3-(phenylthio)benzoic acid − ND ND ND ND ND 4-[(4-bromophenyl)thio]-N- ++/− ND ND ND ND hydroxybutanamide butan-2-yl 4-(phenylthio)butanoate + ++/− ND ND ND PTBA + + +/− ND ND ND methyl 4-[(4- + +/− ND ND ND NDchlorophenyl)thio]butanoate methyl 4-[(4- + + + +/− − NDmethoxyphenyl)thio]butanoate N-hydroxy-4-(phenylthio)butanamide + + +/−ND ND ND propyl 4-(phenylthio)butanoate + + +/− ND ND ND methyl4-(phenylthio)butanoate + + +/− ND ND ND Methyl 4-[(4- + + + − − NDmethylphenyl)thio]butanoate 4-[(4-chlorophenyl)thio]-N- + + +/− ND ND NDhydroxybutanamide N-hydroxy-4-[(4- + + + − − NDmethylphenyl)thio]butanamide 4-[(4-fluorophenyl)thio]-N- + + + +/− − NDhydroxybutanamide methyl 4-[(4- XX + + +/− +/− NDfluorophenyl)thio]butanoate apicidin XX XX  XX¹ +/− − ND TSA XX XX XXXX + + Note1: embryos surviving treatment with 800 nM apicidin (31%, n =36) displayed general toxicity precluding efficacy scoring.

PTBA Analog Efficacy in Renal Progenitor Cells at 3 μM:

Of the 12 compounds that exhibited no significant effect on larvalphenotype, 10 compounds did not expand lhx1a expression as compared withcontrols (no expansion, n=36) (Tables 4 and 5). This group includesHDACis, such as SAHA and tubacin, which exhibit similar potency to TSAin vitro (Bradner, J. E. et al. Chemical phylogenetics of histonedeacetylases. Nat Chem Biol 6, 238-243, (2010)). However, treatment with3 μM Scriptaid or tert-butyl 4-(phenylthio)butanoate increased lhx1aexpression in 39% and 25% of the tested embryos (n=36 each),respectively, as compared with controls (no expansion, n=36) (FIGS. 26,A, C, and E). Therefore, the failure to develop pericardial edemafollowing treatment is a predictive, but not absolute, indicator of anineffective analog.

Lhx1a expression analysis separated the 20 compounds, including PTBA,which caused edemic or lethal phenotypes into four groups. The firstgroup, consisting of 4-(napthalen-2-ylthio)butanoic acid and3-(phenylthio) benzoic acid, caused no expansion of lhx1a in the treatedembryos (Table 5). Therefore, their edemic phenotypes probably resultfrom non-kidney related effects during embryonic development. The secondgroup, consisting of three compounds, expanded lhx1a expression in lessthan 75% of the treated embryos in comparison to controls (FIG. 26A).This group included valproic acid, a known HDACi, (20% expansion, n=35),N-hydroxy-4-[(4-methoxyphenyl)thio]butanamide (31% expansion, n=35), andmethyl 4-[(bromophenyl)thio]butanoate (74% expansion, n=35) (FIGS. 26,B, D, and F). The third group consisted of 12 effective compounds,including PTBA, which expanded lhx1a expression in greater than 90% ofthe treated embryos as compared with controls (Table 3). The eleven PTBAanalogs all contained either hydroxamic or alkylated zinc-bindinggroups. Compounds with these structural motifs also caused the mostedema and lethality in the phenotypic screen (Table 2). Furthermore, allcompounds showing greater than 90% efficacy by in situ hybridizationdemonstrate phenotypic effects similar to or more severe than thosecaused by PTBA (Tables 2 and 3). Therefore, the severity of edemicphenotypes does appear to correlate with the ability of a given PTBAanalog to expand renal progenitor cells with some exceptions aspreviously noted. These compounds were subsequently tested to determinetheir efficacies at sequentially lower concentrations. The final group,consisting of methyl 4-[(4-fluorophenyl)thio)]butanoate, apicidin, andtrichostatin A, killed all treated embryos before they reached the10-somite stage (Table 3). Toxicity of these compounds at 3 μM does notpreclude them from expanding renal progenitor cells at lowerconcentrations. Indeed, treating embryos with 200 nM TSA expands lhx1aexpression in a manner similar to 3 μM PTBA (FIG. 16D compared with FIG.4B). Therefore, the efficacy of these compounds at concentrations below3 μM was assessed along with the previous group.

PTBA analog efficacy in renal progenitor cells at 1.5 μM: Two compounds,apicidin and TSA, were lethal at 1.5 μM, while 13 others demonstratedsome ability to expand lhx1a expression in 10-somite embryos. Two ofthese were partially effective, expanding less than 75% of the treatedembryos when compared with controls (no expansion, n=36) (FIG. 27A).These were 4-[(bromophenyl)thio]-N-hydroxybutanamide (47% expansion,n=36) and methyl 4-[(4-chlorophenyl)thio]butanoate (72% expansion, n=36)(FIGS. 27, B and C). Because they lacked the efficacy of the remaininganalogs, these compounds were not evaluated further. Eleven compounds,including PTBA (83% expansion, n=35) (FIG. 27D), showed efficacy ingreater than 80% of the treated embryos. Four contained hydroxamiczinc-binding groups, with three of these also carrying a substitutedaliphatic cap: N-hydroxy-4-(phenylthio)butanamide (92% expansion, n=36),N-hydroxy-4-[(4-methylphenyl)thio]butanamide (97% expansion, n=33),4-[(4-chlorophenyl)thio]-N-hydroxybutanamide (97% expansion, n=36), and4-[(4-fluorophenyl)thio]-N-hydroxybutanamide (100% expansion, n=36)(FIG. 27, E through H).

Four analogs had methylated zinc-binding groups, with three of thesealso including aliphatic cap substitutions:methyl-4-(phenylthio)butanoate (89% expansion, n=36), methyl4-[(4-methoxyphenyl)thio]butanoate (91% expansion, n=35), methyl4-[(4-methylphenyl)thio]butanoate (100% expansion, n=36), and methyl4-[(4-fluorophenyl)thio]butanoate (100% expansion, n=30) (FIG. 27, Ithrough L). The remaining two analogs demonstrating over 80% efficacycarried propyl- and sec-butyl substitutions, respectively, on theirzinc-binding groups: propyl 4-(phenylthio)butanoate (89% expansion,n=36) and butan-2-yl 4-(phenylthio)butanoate (89% expansion, n=35)(FIGS. 27, M and N). Because each of these 10 PTBA analogs exhibitedefficacy above that of PTBA, these data support the approach oftargeting motifs important in HDACi activity.

PTBA Analog Efficacy on Renal Progenitor Cells at 800 nM:

Treatments at 800 nM revealed two distinct groups of analogs based onefficacy. The partially effective compounds, consisting of PTBA and fiveanalogs, expanded lhx1a expression in less than 25% of treated 10-somiteembryos as compared with controls (Table 3). With the exception of4-[(4-chlorophenyl)thio]-N-hydroxybutanamide, none of these compoundscarried a substituted aliphatic cap. Because of their already limitedefficacy at 800 nM, these compounds were not tested at lowerconcentrations. The five members of the more effective second groupincreased lhx1a expression in greater than 45% of treated embryos ascompared with controls (no expansion, n=36) (FIG. 28A). This group ofanalogs included methyl 4-[(methylphenyl)thio]butanoate (49% expansion,n=35), methyl 4-[(methoxyphenyl)thio]butanoate (57% expansion, n=35),4-[(4-fluorophenyl)thio]-N-hydroxybutanamide (60% expansion, n=35),N-hydroxy-4[(methylphenyl)thio]butanamide (61% expansion, n=36) andmethyl 4-[(4-fluorophenyl)thio]butanoate (64% expansion, n=36) (FIG. 28,B through F).

Each of these compounds contained substitutions in both the zinc-bindinggroup and aliphatic cap. These substitutions represented only a limitedselection of functional groups. The zinc binding groups contained eitherhydroxamic acids or methylated carboxylic acids, while the aliphaticcaps carried either methyl-, methoxy-, or fluoro-substitutions. Theseresults suggest that certain structural motifs impart improved efficacyto the PTBA backbone.

Treatment with 800 nM apicidin killed 69% of the treated embryos (n=36)(Table 3). Surviving embryos exhibited general toxicity that precludedthe scoring of lhx1a expansion (data not shown). TSA treatment at 800 nMwas lethal (Table 3). The five analogs exhibiting greater than 45%efficacy, apicidin, and TSA were retested at 400 nM.

PTBA Analog Efficacy on Renal Progenitor Cells at 400 nM or Below:

Three of the five remaining structural analogs of PTBA and apicidinexhibited a partial ability to expand lhx1a expression at 400 nM incomparison to controls (n=35) (FIG. 29A). Of these, 400 nM4-[(4-fluorophenyl)thio]-N-hydroxybutanamide expanded 35% of treatedembryos (n=34), the highest percentage of any tested PTBA structuralanalog at this concentration (FIG. 29B). Two others, methyl4-[(methoxyphenyl)thio]butanoate (29% expansion, n=35) (FIG. 29C) andmethyl 4-[(4-fluorophenyl)thio]butanoate (26% expansion, n=35),increased lhx1a expression in over 25% of treated embryos. Furthermore,methyl 4-[(4-fluorophenyl)thio]butanoate also caused partial lhx1aexpansion at 200 nM (22% expansion, n=32) (FIG. 29D), while the othertwo structural analogs were ineffective (Table 3). This represents thelowest observed concentration of a structural PTBA analog capable ofexpanding renal progenitor cells.

Treatment with 400 nM apicidin expanded 22% of treated embryos (n=36)(FIG. 29E), but was ineffective at 200 nM. All embryos treated with 400nM trichostatin A died before reaching 10 somites (Table 3). However,treatment with 200 nM TSA caused lhx1a expansion in 100% of treatedembryos as compared with controls (n=29) (Table 3). This observation isin agreement with my previous results (FIG. 16D). Treatment with 100 nMTSA was also effective (97% expansion, n=35), marking the lowest testedconcentration of any PTBA analog, structural or functional, affectingrenal progenitor cells (FIG. 29F).

Toxicity Assays:

The cytotoxicity of the 15 compounds exhibiting efficacy or lethality at3 μM was tested in a conditionally-immortalized mouse podocyte cellline. Podocytes are polarized epithelial cells located on the glomerularbasement membrane that contribute to the integrity of the filtrationbarrier. For the purposes of these experiments, they function as anindicator of renal toxicity. Following 72 hours of treatment with eachcompound concentrations ranging from 30 μM to 3 nM, podocyte viabilitywas assessed by measuring residual metabolic activity (FIG. 30). Fromthese data, the concentration where 50% of the exposed podocytesremained viable (E₅₀) was calculated. Of the 15 analogs tested, onlytwo, TSA and apicidin, caused sufficient cytotoxicity to allow thecalculation of an E₅₀ value. Over the course of three experiments, TSAexhibited a mean E₅₀ of 96 nM (s=51 nM), while the mean E₅₀ for apicidinwas 152 nM (s=54 nM). Treatment with PTBA or each of 12 structuralanalogs never decreased podocyte viability below the 50% threshold, evenwhen tested at 30 μM (FIG. 30). In fact, over three experiments,podocyte viability never dropped 75 below 80% following treatment withany of these compounds. Therefore PTBA analogs, carrying cap and/orzinc-binding group modifications, do not increase the toxicity of thecompound relative to PTBA. These results suggest that the efficacy ofPTBA can be improved through analog development without causing acorresponding increase in compound toxicity.

Methods

Zebrafish husbandry was performed as described above.

Compound Sources and Treatments:

PTBA and methyl-4-(phenylthio)butanoate were synthesized as describedabove. 4-(Naphthalen-2-ylthio)butanoic acid (NSC2733) and3-(phenylthio)benzoic acid (NSC113994), were obtained from the NCI/DTPOpen Repository. APHA compound 8, Apicidin, butanoic acid, PBA,4-phenoxybutanoic acid, 5-phenylpentanoic acid, Scriptaid, TSA, andvalproic acid were obtained from Sigma-Aldrich. MS-275 and SAHA wereobtained from Cayman Chemical Co. 4-(phenylsulfonyl)butanoic acid wasobtained from Matrix Scientific. Tubacin was a gift of Dr. RalphMazitschek of the Broad Institute (Cambridge, Mass.). The remaining PTBAanalogs were synthesized. Three independent groups of 12 chlorinated 2hpf embryos were arrayed in individual wells of 24-well plates. E3medium was removed with a glass pipette and replaced with 800 μItreatment solutions containing 0.5% DMSO in E3 with or without compoundat the reported concentrations.

In Situ Hybridization:

In situ hybridization for lhx1a was performed as described above.Embryos were considered expanded if they exhibited lhx1a expressionconsistent with that resulting from treatment with 3 μM PTBA. Compoundswere classified as effective, partially-effective or ineffective basedon comparison to the effects of PTBA at a given concentration.

Phenotypic Screening:

Phenotypic screening of PTBA analogs was performed identically to theconcentration-response experiments detailed above. Embryos were treatedwith each PTBA analog from 2 to 72 hpf as described above.

Podocyte Cytotoxicity Assays:

The isolation and characterization of conditionally-immortalized mousepodocyte cell line have been previously described (Mundel, P. et al.Rearrangements of the cytoskeleton and cell contacts induce processformation during differentiation of conditionally immortalized mousepodocyte cell lines. Exp Cell Res 236, 248-258, (1997) and Schwartz, E.J. et al. Human immunodeficiency virus-1 induces loss of contactinhibition in podocytes. J Am Soc Nephrol 12, 1677-1684, (2001)). Mousepodocytes were plated at a density of 6,000 cells per 200 μl in 96-wellplates to elicit log-phase growth. The indicated PTBA analogs were addedat 30 μM, 10 μM, 1 μM, 300 nM, 100 nM, 30 nM, or 3 nM and incubated at33° C. for 72 hours. The final DMSO concentration was maintained at 0.2%in all treatments except 30 μM (0.6%). After incubation, cytotoxicitywas analyzed using the Cell Titer-Blue Cell Viability Assay (Promega)per manufacturer's instructions. Reagent (20 μl) was added to 100 μl ofcells and plates were incubated for 2 hours at 37° C. Fluorescence wasread at 79 560Ex/590Em on a Gemini SpectramaxXS (Molecular Devices)plate reader. Viability was calculated as a percentage of the DMSOcontrol, which was considered 100% viability. Data represent the resultsof three independent experiments using duplicate wells for eachcondition. E50 values were determined from the transformed andnormalized data by non-linear regression.

Discussion

From a screen of almost 2000 small molecules, we identified a compound,PTBA, that had not been previously reported as a “hit” in 136 previouschemical library screens (NCBI—PubChem). The success of our screenvalidates the use of an edemic phenotype as an indicator of aberrantkidney development. Furthermore, it emphasizes the importance of soundexperimental design in determining the desired outcome. Many factorsshould be considered before beginning to interrogate librariescontaining thousands of small molecules. Indeed, depending on thepredetermined goals and parameters of the screen, investigators cangenerate completely different data sets using the same compound library.To illustrate this point, chemical screens performed by two labs (deGroh, E. D. et al. Inhibition of histone deacetylase expands the renalprogenitor cell population. J Am Soc Nephrol 21, 794-802, (2010) andMolina, G. et al. Zebrafish chemical screening reveals an inhibitor ofDusp6 that expands cardiac cell lineages. Nat Chem Biol 5, 680-687,(2009)). Both groups tested zebrafish embryos at 10 μM using NCI/DTPDiversity Set compounds drawn from daughter plates derived from the sameDMSO stocks. Furthermore, each lab identified a compound, PTBA or BCI,which expanded progenitor cells, leading to increased kidney or heartsize, respectively. However, the differing experimental approachesutilized by the two groups masked the effects of the other smallmolecule. BCI was identified by treating transgenic embryos carrying afluorescent FGF signaling reporter from 24 to 32 hpf. Since PTBA losesefficacy at about 15 hpf and edema typically does not develop until atleast 48 hpf, PTBA-treated embryos would appear wild-type. Therefore,even if they had recorded interesting secondary phenotypes beyond thoseinvolved in FGF signaling, PTBA would have been missed using thatapproach. Likewise, BCI-treated 72 hpf larvae were scored as wild-typein our phenotypic screen, even though they almost certainly containedenlarged hearts. Although both screens were ultimately successful, theirunique observations depended on the selection of treatment windows andphenotypes appropriate for the research. Without these considerations,the effects of interesting small molecules can be easily overlooked.

Another important factor contributing to the discovery of PTBA was theconcentration chosen for the screen. In this, we were very fortunate.All compounds were ostensibly tested at 10 μM in embryo medium. Had thistruly been the case, the data suggest that PTBA treatment would havekilled all the embryos before reaching 72 hpf. Since we performed nofurther characterization on compounds found to be lethal at thescreening concentration, PTBA might never have been characterized.Luckily, compound concentrations in DMSO stocks can vary several foldfrom the reported value in small molecule libraries (Popa-Burke, I. G.et al. Streamlined system for purifying and quantifying a diverselibrary of compounds and the effect of compound concentrationmeasurements on the accurate interpretation of biological assay results.Anal Chem 76, 7278-7287, (2004)). Thus, the concentration in thePTBA-containing well was probably much closer to 3-5 μM, allowing edemaformation and generating a positive hit. Furthermore, had we decided toscreen at a lower concentration, the larvae may have appeared wild-type,again precluding any further testing. This exposes a flaw in the waymany small molecule screens are performed: very few involve screening atmultiple concentrations. It could be argued that repeated screening of alibrary is a waste of time and effort, especially if hits have beenidentified. However, small molecule libraries generally alreadyrepresent a significant investment of resources, and follow-up screensat different concentrations merit consideration. At the very least,compounds observed to be lethal in the initial screen could be retestedat lower concentrations in search of relevant phenotypes.

Treating zebrafish embryos with PTBA stimulates RA signaling, asevidenced by its effects on the RA-responsive genes cyp26a1 and cmlc2.Consequently, blocking RA signaling at the receptor level withdominant-negative RARα greatly reduced PTBA efficacy. Therefore, PTBAlikely interacts with some elements of the RA signaling pathway in orderto stimulate renal progenitor cell proliferation during pronephricdevelopment. The characterization of PTBA as a novel HDACi suggests thatthe targets are most likely the HDACs controlling the repression ofRA-responsive genes.

In the absence of RA, an RAR/RXR dimer binds the RARE in regulatedpromoters and recruits complexes of corepressor proteins, including anHDAC (FIG. 31A). The HDAC deacetylates nearby nucleosomes, causingchromatin condensation which inhibits gene transcription. When RA bindsthe RAR/RXR dimer, it elicits a conformational change that removes theHDAC from close proximity to the nucleosomes (FIG. 31B). Thisfacilitates the activity of coactivators, such as histoneacetyltransferases, which decondense the chromatin and allowtranscription to occur. By inhibiting the corepressor HDAC, HDACis havebeen hypothesized to lower the RA concentration necessary to activatethe RAR/RXR dimer (FIG. 31C). In this way, PTBA could stimulate RAsignaling at the receptor level without affecting the endogenous RAconcentration.

The downstream target of RA-signaling that mediates the effects of PTBAon renal progenitor cells remains unknown. Since my results argue thatPTBA treatment stimulates renal progenitor cell proliferation withoutsignificantly transforming juxtaposed tissues, the effect is probablylocal. Therefore, PTBA may enhance RA signaling directly within renalprogenitor cells, leading to increased proliferation. Becauseattenuation does not require the synthesis of protein intermediates,this hypothesis is compatible with the previous work of Cartry andcoworkers (Cartry, J. et al. Retinoic acid signalling is required forspecification of pronephric cell fate. Dev Biol 299, 35-51, (2006)).They observed that treating Xenopus embryos with RA caused lhx1aexpansion even in the presence of the protein synthesis inhibitor,cycloheximide. Furthermore, treating kidney epithelial cell lines withRA increased both thymidine uptake and the proportion of cells inS-phase (Anderson, R. J., et al. Retinoic acid regulation of renaltubular epithelial and vascular smooth muscle cell function. J Am SocNephrol 9, 773-781, (1998) and Argiles, A., et al. Retinoic acid affectsthe cell cycle and increases total protein content in epithelial cells.Kidney Int 36, 954-959, (1989)). Therefore, it is possible that theproliferative machinery in renal progenitor cells may respond toRA-signaling in a similar manner.

Therapeutic Potential of PTBA.

In many respects, the process of kidney regeneration mimics that ofnephrogenesis. In both cases, a multipotent cell first proliferates toprovide the necessary raw material and then differentiates into therequired tissue type. Several hypotheses have been proposed to explainthe source of the multipotent cells involved in regeneration. The first,and most generally accepted, suggests that these cells arise from tissuededifferentiation near the site of damage. Other groups argue that themultipotent cells are actually stem cells of intrarenal or extrarenalorigin. In any case, it is reasonable to assume that these cells shareelements in common with renal progenitor cells. Therefore, it washypothesized that they may also exhibit similar proliferation inresponse to PTBA treatment. This hypothesis may explain the observationsmade by other groups regarding the relationship between HDAC inhibitionand renal regeneration. In one study, Imai and coworkers demonstratedthat treating mice with daily injections of TSA attenuated renal damagefollowing injury (Imai, N. et al. Inhibition of histone deacetylaseactivates side population cells in kidney and partially reverses chronicrenal injury. Stem Cells 25, 2469-2475, (2007)). Since PTBA and TSA bothfunction as HDACis and expand renal progenitor cells, they may also actsimilarly in facilitating renal regeneration. In another study, Marumoand coworkers demonstrated that the expression of Hdac5 wassignificantly decreased following acute ischemic damage in mouse kidneys(Marumo, T., et al. Epigenetic regulation of BMP7 in the regenerativeresponse to ischemia. J Am Soc Nephrol 19, 1311-1320, (2008)). Thissuggests that the reduction of HDAC activity may serve as an importantpart of the regeneration process. Therefore, at least some evidencesupports the idea that PTBA may function as a useful therapeuticfollowing acute kidney injury.

To test this hypothesis, we used a transgenic mouse model of acutekidney injury using diphtheria toxin (DT) as a nephrotoxic agent. Unlikehumans, mice are normally resistant to DT exposure. This resistancearises from several amino acid differences in the protein acting as theDT receptor, heparin-binding EGF-like growth factor (Hbegf) (Mitamura,T., et al. Diphtheria toxin binds to the epidermal growth factor(EGF)-like domain of human heparin-binding EGF-like growthfactor/diphtheria toxin receptor and inhibits specifically its mitogenicactivity. J Biol Chem 270, 1015-1019, (1995)). Transgenic mice (PTC-DTR)express human HBEGF in their proximal tubules, creating the conditionsfor an acute and specific damage event. Since blood is a bufferedsolution, testing PTBA would have been a poor choice. At neutral pH, thezinc-binding group would be deprotonated and the resulting polar chargewould greatly decrease its absorption through cell membranes. Instead wetested an effective structural analog carrying a methylated zinc-bindinggroup, methyl 4-(phenylthio)butanoate (MPTB). PTC-DTR mice were injectedwith DT and damage was allowed to accrue for one day. At this time, micewere given daily injections of MPTB or a DMSO control for the next sixdays. Preliminary data from a single group of mice suggest that MPTBtreatment significantly increases the rate of renal recovery byapproximately 30% (FIG. 32). Therefore, PTBA analogs demonstrate somepromise as renal therapeutics following acute kidney injury.

Toxicity is also an important consideration during the development ofany drug. At its effective concentration, PTBA exhibited much mildereffects on juxtaposed mesodermal tissues in comparison to TSA.Furthermore, structural PTBA analogs, even when administered at 30 μM,demonstrated little effect on podocyte cell viability in cell cultureassays. However, the data above suggests that treating podocytes with100 nM TSA would be expected to kill at least 50% of the cells within 72hours. There are at least two possible explanations for this differencein toxicity, each reflecting different considerations of HDAC-HDACibinding. Crystallography studies have revealed how several HDACis blocksubstrate access by interacting with the Zn²⁺ in the catalytic site ofan HDAC. Therefore, most HDACis, including carboxylic and hydroxamicacids, function as competitive inhibitors of HDACs, althoughnoncompetitive HDACis, such as Trapoxin A, have been characterized. Inthis example, taken from the Protein Data Bank, TSA occupies the bindingsite of human HDAC7 by coordinating its hydroxamic acid motif with thecatalytic Zn²⁺ (FIG. 33). In general, hydroxamic HDACis bind HDACs muchmore strongly than HDACis containing carboxylic acid zinc-bindinggroups, such as PTBA (Jacobsen, F. E., et al. Chem Med Chem 2, 152-171,(2007)). This is because PTBA should form only one coordinate bond withthe active site zinc, an arrangement known as monodentate binding. Incontrast, the hydroxamic acid of TSA binds in a bidentate fashion,forming two coordinate bonds with the active site zinc. This differenceis reflected in the results of our in vitro HDAC analysis. TSA isobserved to inhibit all HDAC activity at micromolar concentrations,while PTBA reduced HDAC activity by only 70% at millimolarconcentrations. However, this large efficacy difference is not evidentin vivo. PTBA and TSA were found to generate similar increases inhistone hyperacetylation at concentrations within about one log of eachother. These data suggest that additional factors may be involved indetermining compound efficacy in the context of a whole organism.

The difference in HDAC isoform specificity between carboxylic andhydroxamic acids may provide a possible explanation for theseobservations. HDAC isoforms are separated into four classes based ontheir size, homology, cellular localization, and catalytic activity(FIG. 34). For example, Class I isoforms generally exhibit nuclearlocalization, while Class II HDACs are primarily cytoplasmic(Balasubramanian, S., et al. Isoform-specific histone deacetylaseinhibitors: the next step? Cancer Lett 280, 211-221, (2009)). Carboxylicacid HDACis are considered to be specific inhibitors of Class I and IIaHDACs, while hydroxamic acids target Class I, II, and IV isoforms (FIG.34, Bieliauskas, A. V. et al. Isoform-selective histone deacetylaseinhibitors. Chem Soc Rev 37, 1402-1413, (2008); Bolden, J. E., et al.Anticancer activities of histone deacetylase inhibitors. Nat Rev DrugDiscov 5, 769-784, (2006); Butler, K. V. et al. Chemical origins ofisoform selectivity in histone deacetylase inhibitors. Curr Pharm Des14, 505-528, (2008); and Khan, N. et al. Determination of the class andisoform selectivity of small-molecule histone deacetylase inhibitors.Biochem J 409, 581-589, (2008)). Meanwhile, the Class III HDACs(sirtuins), utilize an NAD+-dependent catalysis mechanism that remainsunaffected by carboxylic or hydroxamic HDACis (FIG. 34). Although theclass specificity of PTBA has not yet been confirmed, it can behypothesized to function as a Class I/Ha-specific inhibitor like othercarboxylic acids.

Several inferences can be made from these isoform specificities. First,if PTBA efficacy in zebrafish embryos does indeed require the inhibitionof one or more HDAC isoforms, then hdacs 6, 10, and 11 are probably nottargeted. Therefore, it is possible that the increased toxicity observedin TSA-treated embryos may result from its effect on these hdacs. Ofparticular interest is HDAC6, which functions as amicrotubule-associated deacetylase (Hubbert, C. et al. HDAC6 is amicrotubule-associated deacetylase. Nature 417, 455-458, (2002)).Affecting the posttranslational modifications of the microtubule networkcauses broad effects on cell signaling and the maintenance ofhomeostasis. However, embryos treated with tubacin, an HDAC6-specificHDACi (Haggarty, S. J., et al. Domain-selective small-molecule inhibitorof histone deacetylase 6 (HDAC6)-mediated tubulin deacetylation. ProcNatl Acad Sci USA 100, 4389-4394, (2003)), appeared wild-type in myphenotypic screens. It is possible that tubacin's Log P of 6.34, thehighest of any tested PTBA analog, may prevent efficient compoundabsorption and could account for the lack of phenotype. Furthermore, thebroad specificity of hydroxamic HDACis suggested that hydroxamic PTBAanalogs would demonstrate greater efficacy at the expense of increasedtoxicity. Indeed, several hydroxamic analogs increased lhx1a expressionin treated embryos at concentrations equal to or less than PTBA.However, the at a from zebrafish embryos and podocyte culture suggestthat this modification has little to no effect on compound toxicity.This may imply that the hydroxamic analogs remain weak inhibitorsdespite the improved strength of the zinc-binding group. Accordingly,their HDACi activity should be determined empirically in future in vitroassays. Alternatively, the products generated from the metabolism of TSAmay cause the deleterious effects observed in treated embryos. Indeed,some evidence suggests that the N-demethylated byproducts of TSA remainpharmacologically active and persist in the circulation for at least anhour following administration (Sanderson, L. et al. Plasmapharmacokinetics and metabolism of the histone deacetylase inhibitortrichostatin after intraperitoneal administration to mice. Drug MetabDispos 32, 1132-1138, (2004)). However, it is currently unknown if thesemetabolic waste products affect normal cellular processes.

Previous clinical studies with known HDACis suggest that structuralanalogs of PTBA may function as viable therapeutics. Indeed, bothtrichostatin A (Vorinostat) and valproic acid have received U.S. Foodand Drug Administration (FDA) approval for oral administration to treatT-cell lymphoma and epilepsy, respectively. Importantly, the sodium saltof PBA (Buphenyl), one of the closest structural analogs of PTBA, alsoreceived FDA approval in 1996 for the treatment of urea cycle disorders.To date, Buphenyl remains the only FDA-approved treatment for chronicmanagement of the excess blood ammonia (hyperammonemia) generated byenzymatic deficiencies in urea metabolism. Prescribed daily doses ofBuphenyl as an oral medication often reach gram quantities, with somepatients taking as much as 20 g/day. Thus, it may be possible to safelyadminister other PTBA analogs at similarly high doses. In addition,Buphenyl exhibits good bioavailability of about 80% when taken orally.Unfortunately, Buphenyl has a short half-life (t_(1/2)˜1 hr) followingadministration, as it is rapidly converted to phenylacetic acid.However, this limitation can be easily overcome by prescribing multipledaily dosings. Therefore, the favorable pharmacological properties ofsodium PBA support the future development of other PTBA analogs forclinical use.

Certain questions remain open from this study. Although, the zebrafishpronephros serves as an excellent model of individual metanephricnephrons, metanephroi are complex organs consisting of millions ofnephrons. It is unknown how accurately simple pronephric models reflectconditions within the broader context of general metanephric kidneyfunction. Future experiments with PTBA analogs in mouse models of kidneyinjury may help to address this. Furthermore, while my data suggest thatPTBA treatment affects normal pronephric function, thus causing theedemic phenotype, this was not determined empirically. Although thepronephros is almost certainly nonfunctional at 48 hpf because theglomerulus has not yet formed, fluorescent dextran injections couldprovide further confirmation. Indeed, it would be interesting todetermine if PTBA-treated embryos manage to form functional pronephroilater in development. This research has provided some insight into themechanism of PTBA efficacy, revealing the involvement of the RAsignaling pathway and HDAC inhibition. However, the specific target ofPTBA activity remains unknown. Ostensibly, this should be an HDACisoform or isoforms, perhaps representing a specific HDAC class.However, it is important to note that HDACis modify many non-histoneproteins in their capacity as lysine deacetylases (Buchwald, M., et al.HDACi-targets beyond chromatin. Cancer Lett 280, 160-167, (2009)). Thesetargets include hormone receptors, signal transducers, transcriptionsfactors, structural proteins, and chaperones. Therefore, targetidentification may not be a straightforward prospect. However, aninvestigation of possible HDAC targets still represents the best placeto start. These preliminary results suggest that the expression patternsof individual HDAC isoforms remain relatively ubiquitous in 24 hpfzebrafish embryos (data not shown). It may be helpful to determine ifany of these become compartmentalized in the pronephric region later indevelopment. These isoforms would be good candidates as potential PTBAeffectors. Furthermore, like other carboxylic acid HDACis, PTBA would beexpected to exhibit some Class I/IIa specificity. If this characteristicis confirmed in future investigations, then isoforms of these classesshould also merit some interest. Morpholino knockdown of thesecandidates in an effort to recapitulate lhx1a expansion in the IM wouldprovide an excellent approach for target validation. When developingpotential human therapeutics using zebrafish, consideration should begiven to the variation in HDAC orthologs across species. Althoughzebrafish hdacs, such as hdac1, share a large degree of amino acididentity with the HDACs of other vertebrates, discrepancies remain (FIG.35). These differences could alter the efficacy of PTBA or its analogswhen applied in other model systems or in humans. Encouragingly, PTBAdemonstrated the ability to function in vitro as an HDACi using humanHDAC isoforms derived from HeLa cell extracts. However, this result doesnot eliminate the possibility of differing efficacies when assessingindividual HDAC orthologs.

In addition to the points raised above, several other open questionsremain unanswered. Potent HDACis, such as SAHA and MS-275, showed noeffect on zebrafish embryos when they were treated at 3 μM. This was anunexpected result which could reflect poor absorption of the compoundsinto the embryos, short half-lives, or a zebrafish-specific effect.Hyperacetylation assays could be performed to determine if the compoundsare indeed able to exert a physiological effect on embryos, despite thelack of edemic phenotype. Furthermore, exploring the downstream effectsof HDAC inhibition, such as the stimulation of coactivators, wouldprovide further confirmation of the mechanism of PTBA efficacy. Forexample, pharmacologically inhibiting the activity of histoneacetyltransferases (HATs) would be expected to block PTBA efficacy byinterfering with its signaling. One final point of interest is betterunderstanding the differences in PTBA efficacy when compared to TSA inthe in vitro and in vivo assays. In vitro, TSA is thousands of timesmore effective than PTBA in inhibiting HDAC activity, while in vivo thedifference is greatly reduced. Of course, the varying HDAC classspecificities of the two compounds may play a role in this effect.However, the structural elements of PTBA, particularly the thiol group,is suspected to provide the compound with unique in vivopharmacokinetics.

These results demonstrate that several PTBA analogs exhibit improvedefficacy with little to no increase in toxicity. These second generationcompounds are excellent candidates to improve renal recovery in futureanimal studies. However, this work marks only the first steps in thedevelopment of this family of compounds. Using only simple functionalgroup substitutions, we were able to identify PTBA structural analogsthat were only several times less effective than TSA. However, thesesame analogs exhibit several hundred times less toxicity than TSA inpodocyte cell culture. This represents the key benefit offered by thisclass of “weak” inhibitors. It is true that almost all of the effectivePTBA analogs cause some degree of edemic and/or lethal phenotypes duringzebrafish development. However, these teratogenic effects should not bean issue when treating an adult, whose tissues primarily consist ofdifferentiated cells. Indeed, as shown below, PTC-DTR mice treated withm4PTB exhibit no overt signs of toxicity (data not shown). Because thesecond generation PTBA analogs exhibit little toxicity, there is stillample room for further efficacy improvements. The results of my researchoffer some guidelines for this process. In general, substitutions of thealiphatic cap region increased compound efficacy. Adding functionalgroups to the phenyl ring represents a simple way to increase the Log Pvalue of the base compound, which may aid absorption through biologicalmembranes. Alternatively, the cap substitutions may interact with aminoacids in the binding pocket of the HDAC, improving the affinity of thecompound. Interestingly, even halogenated groups, which are oftenmetabolized into toxic byproducts (Pohl, L. R. et al. Electrophilichalogens as potentially toxic metabolites of halogenated compounds.Trends Pharmacol Sci 5, 61-64, (1984)), appear to be tolerable. Thesulfur atom forming the connecting unit of PTBA also appears to be anactivity determinant. In comparison to its substituted analogs, onlyPTBA exhibited significant efficacy. Since the Log P values of PTBA andthe analogs carrying connecting unit substitutions are similar, it isdoubtful that improved absorption plays a role. More likely, the sulfuratom forms unique interactions within the HDAC binding pocket. This mayinclude serving as a hydrogen bond acceptor due to the presence of anunbound electron pair.

Unfortunately, only one analog carrying a linker group was available fortesting. Adding a bulky ring structure to the linker decreased theanalog's efficacy relative to PTBA. The increased rigidity of thenormally flexible hydrophobic chain may explain this effect. It isbelieved that increasing the length of the linker region remains anuntapped opportunity to generate a better analog. Indeed, the linkers ofthe more potent HDACis, including TSA and SAHA, contain seven and eightcarbons respectively. Finally, analogs carrying methylated or hydroxamiczinc-binding groups exhibit improved efficacy. Adding alkyl groups tothe zinc-binding group might be expected to interfere with the formationof coordinate bonds with the active site zinc. However, it is likelythat the ubiquitous esterases found in target cells hydrolyze and removethe alkyl groups upon absorption. Furthermore, since alkylation preventsdeprotonization of the carboxylic acid, these analogs would be expectedto be more effective in buffered solutions. The hydroxamic analogs ofPTBA also demonstrated improved efficacy, although perhaps not as muchas expected. Although they possess stronger zinc-binding groups, theysuffer from increased polarity as evidenced by lower Log P values.Therefore, the expected increase in binding affinity may becounterbalanced by decreased absorption. Importantly, the hydroxamicPTBA analogs demonstrate none of the toxicity of TSA in cell cultureexperiments. Further modifications of these structures, perhapsincorporating some elements of the TSA structure, may yield a family ofhighly potent compounds.

Example 4

Initial studies, above, used a phenotypic marker of the ability toexpand the renal progenitor cell field in larval zebrafish, andidentified the first member of a novel class of HDAC inhibitors, PTBA aswell as a few structural and functional analogs. These findings form thebasis for further study. However, this technology is limited as it isdependent on subjective interpretation of a subtle phenotypic change inthe fish. Therefore, in order to develop a more objective and highercontent screen, we developed an image-based high-content screen usingtransgenic zebrafish (Vogt A, et al. Automated image-based phenotypicanalysis in zebrafish embryos. Dev Dyn. 2009; 238(3):656-63). For thiswe first generated transgenic cad17:EGFP zebrafish which express GFP inrenal tubular epithelium. By treating zebrafish embryos and allowingthem to develop into larvae, we can use Tg(Cad17:EGFP) zebrafish toidentify expansion of the renal field. Tg(Cad17:EGFP) zebrafish is atransgenic line that was prepared by isolation of an approximately 5 kb(5066 bp) promoter element and approximately 3 kb (3221 bp) first intronelement from the cadherin 17 locus, cloning it into a plasmid along withthe enhanced GFP open reading frame and injecting if into zebrafishembryos to established transgenic lines using the meganucleasetransgenesis method (Soroldoni et al., Simple and efficient transgenesiswith meganuclease constructs in zebrafish, Methods Mol Bio 2009; 546:117-30.).

Exploiting an object-oriented image analysis methodology that modelshuman cognitive processes, termed Cognition Network Technology (CNT)(Vogt A, et al. Dev Dyn. 2009; 238(3):656-63 and Vogt A, et al.High-content analysis of cancer-cell-specific apoptosis and inhibitionof in vivo angiogenesis by synthetic (−)-pironetin and analogs. ChemBiol Drug Des. 2009; 74(4):358-68), we have been able to quantify thesize of the fluorescently labeled kidney field in embryos treated withour lead PTBA analogue, m4PTB (FIG. 36). Structure assignment isachieved by detection of regions within the image based on features suchas brightness, size, and shape. What permits the identification ofbiologically meaningful domains (head, tail, yolk, tubules) from thoseinitial features is that a human observer immediately assignscontext-specific information to objects based on their knowledge oflarval morphology. CNT provides this context by permitting users toassign relational information to image objects through context-dependentclassification and segmentation to produce a network of objects andrelationships. The method is independent of imaging platform, imageformat, and phenotype.

Primary and Secondary Zebrafish Screens:

The goal of these studies is to evaluate PTBA analogues with improvedactivity/toxicity characteristics. Since the lead compound, PTBA, is acarboxylic acid, there are potential stability issues in bufferedenvironments such as blood. It is also desirable to utilize compoundsthat have effective dose ranges in the nano-molar range, as these aremore likely to be useful in vivo. As described above, several PTBAanalogs were synthesized, including methyl, n-propyl, and sec-butylPTBA. The methyl PTBA (m4PTB) analogue, demonstrated an effective dosefor the renal progenitor assay in the 800 nM range, and it is shown tobe effective in accelerating functional recovery from AKI (see FIG. 39).The efficacy of the new PTBA derivatives relative to that of m4PTB isbeing evaluated, and those compounds with equal or greater activity ofm4PTB can be used in a next phase of the screen.

Considerations in Designing and Synthesizing New PTBA Analogues:

Esterified compounds, such as m4PTB offer desirable characteristics forabsorption and distribution in mammals, providing a first avenue ofpursuit in analog building. Commercially available thiophenols (i.e.,substituted on the phenyl or naphthyl rings) are prepared in order todetermine the constraints of electron density and bulk on the aromaticregion. A short series of esters designed to extend the biologicallifetime of PTBA are prepared (i.e., ethyl, n-propyl, iso-propyl,n-butyl, sec-butyl and tert-butyl). All of these are easily achievableat the gram scale. Two mixed esters of PTBA (and any activephenyl-substituted or naphthyl analogues) are prepared. For the reverseester, valproic acid ethyl ester is reduced to its alcohol, which isthen used to esterify PTBA. Some of these esters contain a chiral carbonin the alcohol-derived portion of the compound (e.g., sec-butyl,valproyl and valproate). Those esters with such chirality, if active,are separated into their enantiomers by HPLC or supercritical fluidchromatography (SFC) using standard techniques and the enatiomers testedfor activity. Finally, the free acid forms of PTBA and its phenylring-substituted/expanded analogues are converted to their hydroxamideforms via activation of the acid moiety and reaction with hydroxylamine.Based on this combinatorial chemistry, we expect to generate ˜200 PTBAanalogues for screening using the zebrafish reporter assay. Compoundsthat prove effective are scaled up for the mouse studies.

Considerations in Screening New Analogues for Renal Progenitor CellExpansion:

For all second-generation compounds, the cad17:EGFP transgene is used asthe primary readout to assay for expansion of kidney field (FIG. 36).PTBA analogues that expand cad17:EGFP expression are subjected to asecondary screen. The secondary screen utilizes two lines,Tg(lhx1a:EGFP) (Swanhart et al., 2010 Characterization of an lhx1atransgenic reporter in zebrafish, Int J Dev Biol 54(4): 731-6) andTg(pax2a:GFP)(Picker et al., A novel positive transcriptional feedbackloop in midbrain-hindbrain boundary development is revealed throughanalysis of the zebrafish pax2.1 promoter in transgenic lines,Development 2002 129 (13): 3227-39). The lhx1a:EGFP and pax2a:GFPtransgenic lines are expressed in renal progenitor cells (see above),and allows confirmation that expansion of the nephric field seen withcad17:EGFP is the result of expansion of renal progenitor cells. Thedescribed analog families are expected to result in more than 200 newcompounds. While we can handle all the compound dose curves in thelhx1a:EGFP and pax2a:GFP reporter assay, we can employ the Toplissapproach for analogue synthesis to initially narrow our focus (Martin YC, et al. Examination of the utility of the Topliss schemes for analogsynthesis. J Med Chem. 1973; 16(5):578-9). Dosage curves for each newcompound are added to Tg(cad17:EGFP) embryos starting an 3 uM as thehigh dose and using a semi-log curve (1 uM, 600 nM, 300 nM, 60 nM, 30nM) and assayed for expansion of the nephric mesoderm. Since m4PTB isresponsive in the 800 nM range, we focus on second-generation compoundsthat have lower effective dose. Such compounds are assessed in targetefficacy and toxicity assays.

Efficacy and Toxicity Studies in Mice:

Candidate PTBA analogues are subjected to general toxicity and efficacystudies in mice.

Efficacy Studies:

PTBA analogues are soluble in 1% DMSO/water, are administeredsubcutaneously as a single injection in adult CD1 mice, and kidneysharvested at 12, 24 and 48 hours to evaluate histone acetylation (whichshould increase with HDACi). PTBA analogs are used at a dose rangedetermined from comparative dose efficacy with m4PTB in the secondaryzebrafish screen. Acetylated Histone H3 K9 (Ac-H3 K9) is evaluated, asthis is most easily detected by Western blot in nuclear extracts frommouse renal tissues. Preliminary studies compared the effect ofTrichostatin A (an established, but toxic HDACi) at an effective dose inthe zebrafish screen (0.2 mM) with an equivalent dose of m4PTB (0.8 mM),and then ×2 and ×4 dose increments (molar concentration in vivo based onestimate uniform distribution relative to body mass of water). 3.4 mg/kg(4×) m4PTB induced maximal expression of Ac-H3 K9 after 24 hours (FIG.37). This effect was increased compared with TSA or lower doses ofm4PTB, so we elected to use this dose of m4PTB (3.4 mg/kg) for AKIstudies in mice. For definitive studies, we can compare 1×, 2× and 4×equivalent doses of each new PTBA analogue with 3.4 mg/kg m4PTB, andevaluate renal Ac-H3 K9 at the indicated time points. Studies may beperformed on 3 mice/group.

Toxicity Studies:

Having established an effective dose regimen for the PTBA analogue,cumulative toxicity studies are performed in mice over a 7-day period(the duration of treatment). Compound dosages are based on efficacystudies, and only those compounds behaving as well or better than m4PTBare evaluated. Vehicle controls are compared with 3 doses of thecompound given at the indicated time intervals (log scale, first dosebased on efficacy studies). Mice are sacrificed after 7 days and undergonecropsy and tissue analysis for histological evidence of organtoxicity. In addition renal function (BUN and creatinine), liverfunction (AST, ALT, ALP, Bilirubin and Albumin) and CBC (WBC, Hb andplatelets) from terminal blood samples are evaluated. LFT and CBC areperformed through a commercial lab. Studies are performed in 7 mice pertreatment limb. Only those compounds showing little to no evidence oftoxicity within a log of their effective dosage are further evaluated.

Example 5—the Effect of Second-Generation HDACi on Functional Recoveryfrom AKI in Mice

PTC-DTR Model of AKI:

We have developed a mouse model of AKI that is ideally suited for ourprimary screening studies. Our experience with the more establishedmodels (ischemia/reperfusion, HgCl₂ and cisplatin) has shown that thereis significant variability between mice such that functional studieshave to be performed with 10-15 mice per group. Transgenic mice expressthe diphtheria toxin (DT) receptor, human HB-EGF, in proximal tubularepithelial cells (PTC) using a 2.2 kb fragment of the proximal gGT-1promotor. Characterization of transgene expression indicates thathHB-EGF is only expressed in PTCs (>95% of cells, data not shown). Asingle IP injection of DT causes AKI in transgenic but not wild typemice, with dose-dependent PTC injury and increased blood urea nitrogen(BUN, a measure of renal function) that peaks between days 3 and 4 andreturns to baseline by day 7 (FIG. 38). Male transgenic mice are moresensitive to DT than female mice, which show a milder and slightly laterpeak in BUN (see FIG. 39). However, we find reproducibly tight datapoints in both female and male mice using 5-6 mice per group. Moreover,while BUN returns to baseline after 7 days, there is renal fibrosis athigher doses of DT (FIG. 38, panel D). The latter finding is importantsince the purpose of this screen is to identify compounds thataccelerate tubular regeneration following injury, and in so doingprevent long-term fibrotic sequelae resulting from severe AKI, which isbeing recognized increasingly as a cause of chronic renal insufficiency.

We evaluated efficacy and effective dosage of a second-generation PTBAanalogue, m4PTB, by determining the kinetics of renal Histone H3 K9acetylation (H3K9Ac) following a single SC injection. We saw maximalinduction of H3K9A at 3.4 mg/kg, which persisted for 24 hours (FIG. 37).Therefore, using the PTC-DTR model, we treated female transgenic micewith 3.4 mg/kg m4PTB (or 1% DMSO vehicle) 24 hours after injection withDT (0.1 mg/kg, a dose of that induces relatively mild AKI), andcontinued with daily m4PTB (or control) injections for 6 days. There wasa significant, 30% reduction in peak BUN 4 days after injury in them4PTB group (FIG. 39). Moreover, while further studies are beingperformed to determine: 1) whether m4PTB is efficacious in more severeDT-induced AKI; and 2) whether accelerated renal recovery from moresevere AKI is associated with reduced renal fibrosis at 4 week, thesefindings establish m4PTB as our first lead compound for further testingin mouse models of AKI.

Studies performed in male PTC-DTR mice using two different doses of DTpredicted to induce mild (0.1 mg/kg) and severe AKI (1 mg/kg) (see FIG.38), determine whether PTBA analogues have beneficial effect in mild,severe AKI or both. Treatment with the PTBA analogue is initiated 24hours after DT injection and continued daily thereafter for 7 days. Eachstudy includes 3 groups of mice: 1% DMSO (vehicle), m4PTB at 3.4mg/kg/day and the new analogue at the dose established from renalhistone acetylation studies. Based on our experience using this modelseven mice per group are used. Mice have daily BUN assays. If BUNimproves significantly (as determined by area under the curve growthcurve analysis and post-hoc T-Test with Bonferroni correction), resultsare validated on the same samples by measuring serum creatinine by HPLC(Yuen P S, Dunn S R, Miyaji T, Yasuda H, Sharma K, Star R A. Asimplified method for HPLC determination of creatinine in mouse serum.Am J Physiol Renal Physiol. 2004; 286(6):F1116-9). In addition tomeasuring BUN, mice are sacrificed at 28 days to evaluate renal fibrosis(trichrome blue and sirius red staining). Quantification of tubularatrophy and fibrotic indices and collagen accumulation from sirius redstaining are quantified using the BIQUANT image analysis system. Theseanalyses determine whether the PTBA analogues also reduce long termrenal fibrosis following AKI. Compounds that accelerate recovery fromAKI as well or better than m4PTB and that also reduce long term fibrosisare moved forward onto the secondary screening studies in mice.

Compounds that accelerate AKI recovery as well or better than m4PTB, andshow beneficial effects on post-injury renal fibrosis in our primaryscreen, will be studied more extensively. This secondary screen willevaluate effects of these compounds using three other models of AKI thatreflect the spectrum of disease pathology in human AKI: 1)ischemia/reperfusion; 2) toxin (cisplatin); and 3) sepsis (caecalligation and perforation). Studies are performed using an outbred strainof mice (CD1) in order to more closely mimic the greater geneticdiversity of patients in clinical settings.

Example 6—Additional Models

Caecal Ligation Model of AK:

mice are treated with second generation PTBA analogues 24 hours afterinjury and outcome determined relative to m4PTB and vehicle. BUN andserum creatinine are evaluated in all the mice, as well as renalhistology for fibrosis on follow up where long term survival is feasible(ischemia/reperfusion and cisplatin models), as outlined above. Formodels showing improved renal function and/or beneficial effects on longterm renal fibrosis, additional cohorts of mice are set up to evaluaterenal histology pre-injury, at day of peak BUN and 7 days post-injuryfor renal tubular injury scores, tubular proliferation (BrdUincorporation) and apoptosis indices (TUNEL assay). Details of thespecific models are outlined below:

Ischemia/Reperfusion (IR) Injury:

IR injury provides a model of AKI that mimics the effects ofhypotension, hypovolemia and aortic surgery on the kidney in humans.Injury occurs in highly metabolically active S3 segment PTCs in theouter stripe of the medulla. The model originally developed in rats hasbeen modified for use in mice (Kennedy S E, Erlich J H. Murine renalischaemia-reperfusion injury. Nephrology (Carlton). 2008; 13(5):390-6)(19).

Cisplatin-Induced AKI:

There are different models of nephrotoxin-induced AKI. Cisplatin hasdirect cytotoxic effects on both proximal and distal tubular epithelium(dominant cortical PTCs) and has been used extensively as a model of AKIin mice and rats. Moreover, cisplatin is a chemotherapeutic agent usedin the treatment of a variety of different malignancies, so the modelhas relevance to a human disorder. Mice injected on two consecutive dayswith 10 mg/kg cisplatin IP develop reproducible and reversible injuryover a 7-day course but with reasonable survival (death occurs from bonemarrow suppression and AKI) (Bi B, Schmitt R, Israilova M, Nishio H,Cantley L G. Stromal cells protect against acute tubular injury via anendocrine effect. J Am Soc Nephrol. 2007; 18(9):2486-96).

Sepsis (Cecal Ligation and Perforation):

Sepsis is one of the most common causes of AKI in humans, and is acontributory factor in the etiology of AKI in many different settings(including hypotensive AKI). One model that reflects the clinicalscenario of sepsis-associated AKI is a surgical model that involvescecal ligation and limited cecal perforation (Doi K, Leelahavanichkul A,Yuen P S, Star R A. Animal models of sepsis and sepsis-induced kidneyinjury. J Clin Invest. 2009; 119(10):2868-78. PMCID: 2752080). Bylimiting the length of the ligated cecum, and treating mice withbroad-spectrum antibiotics and S/C normal saline each day, mice survivefor a period of 5-7 days and reproducibly develop AKI. PTBA analoguetreatments are initiated 24 hours after surgery, as outlined above.

Alternatively, the LPS-induced AKI model in which transient AKI isinduced by infusion of LPS (Doi K, et al. J Clin Invest. 2009;119(10):2868-78. PMCID: 2752080) can be used.

PTBA analogues will accelerate the rate of renal recovery in AKI.However, the efficacy of any single compound-based therapy isnecessarily limited by the maximal biological activity of their class.Since we know that this class of HDACi improves the rate of recoveryfollowing AKI, a complementary approach is to determine if a combinationof compounds is more effective than a PTBA analogue alone. Thiscombinatorial approach is a standard clinical methodology for treatingdisease (Boner G, et al. Combination antihypertensive therapy in thetreatment of diabetic nephropathy. Diabetes Technol Ther. 2002;4(3):313-21) (22). On this basis, by evaluating the effect ofsub-optimal levels of the lead PTBA analogue, m4PTB, in combination witheither (a) FDA approved drugs or (b) drug-like compounds, we expect tofind a combination that can greatly expand the renal progenitor cellpopulation in our zebrafish screen.

Whole-Embryo Combinatorial-Synergism Screen:

We know that m4PTB can cause a measurable expansion of the cad17:EGFPtransgene at dose between 800 nM to 1 μM (FIG. 36). In addition, in anrenal progenitor cell study using the lhx1a:EGFP transgene, we can startto see expansion of the renal progenitor cell population at doses as lowas 600 nM (data not shown). Therefore, a combinatorial-synergism screenis performed by combining 600 nM m4PTB with different compoundlibraries. As with the second-generation compound screen, the cad17:EGFPtransgene is used as the primary readout for expansion of kidney field,and compounds that expand cad17:EGFP expression are subjected to asecondary screen. Those compounds that enhance efficacy of sub-optimaldoses of m4PTB are evaluated for combinatorial drug toxicity andefficacy, before progressing to our primary AKI screen.

Compound Selection:

It is important to seek new structures as well as to improve on theactivity and selectivity of the current inhibitors we have identified.Therefore, (1) collections of FDA approved drugs or agents with knownbiological activity are examined and (2) libraries of maximal chemicaldiversity selected for their drug-like characteristics are interrogated.We have in hand the Library of Pharmacologically Active Compounds(LOPAC), the NCI 400 compound clinical trial set, and the 10,000-memberTimTec ActiProbe library. The LOPAC library is extensively used at theUPDDI and contains 1280 compounds with known biological activity,including FDA approved drugs. The NCI 400 compound clinical trial set ofFDA approved compounds. (Our original compound was found in the NCIdiversity set.) The ActiProbe library was computationally selected byJarvis-Patrick sampling to represent the chemical diversity of a large2,000,000 member compound collection, and is biased towards diversedrug-like molecules through cheminformatics filtering including LipinskiRule parameters.

Screen Outcomes:

We expect to identify a set of compounds that can work in concert withm4PTB to improve post-renal damage recovery. Once a hit is found a dosecurve is performed in combination with m4PTB and individually for thenew hit. Any compounds that expand the renal progenitor cell populationwithout the aid of m4PTB (not an additive effect, but an independenteffect) are just as important as compounds that work in a synergisticmanner. If a large number of hits are identified, those that work incombination with m4PTB are priority compounds for assessing in mousetoxicity and efficacy studies. By using m4PTB, any potential hits can bemoved quickly though the efficacy and toxicity studies into the PTC-DTRAKI model.

Example 7—Synthesis and Evaluation of PTBA Analogs

A number of PTBA analogs were synthesized essentially as shown in FIG.12 and evaluated using a pre-screening method essentially as describedabove. Physical data, including spectral analysis was performed for anumber of the analogs, as indicated below. The following are physicaldata for various synthesized compounds:

Compound: VNK-I-154; Yield: 95%; Rf: 0.10 Hex/EA 50:1; Mp, ° C.:colorless oil; ¹H NMR (CDCl₃, 600 MHz): δ: 0.90 (t, J=7.8 Hz, 3H), 1.21(d, J=6.0 Hz, 3H), 1.50-1.63 (m, 2H, diastereotopic CH₂), 1.97 (quintet,J=7.2 Hz, 2H), 2.46 (t, J=7.2 Hz, 2H), 2.98 (t, J=7.2 Hz, 2H), 4.86(sextet, J=6.6 Hz, 1H), 7.17-7.21 (m, 1H), 7.30 (t, J=7.8 Hz, 2H), 7.36(dd, J=8.4 Hz, J=1.2 Hz, 2H); ¹³C NMR (CDCl₃, 150 MHz): δ: 9.73, 19.49,24.51, 28.80, 32.97, 33.28, 72.29, 126.02, 128.93, 129.30, 136.13,172.62; HPLC/MS: >95%; HRMS: Calcd. 253.1262, Observed 253.1269.

Compound: VNK-I-157; Yield: 56%; Rf: 0.30 Hex/EA 9:1; Mp, ° C.:colorless oil; ¹H NMR (CDCl₃, 600 MHz): δ: 1.46 (s, 3H), 1.94 (quintet,J=7.2 Hz, 2H), 2.39 (t, J=7.2 Hz, 2H), 2.97 (t, J=7.2 Hz, 2H), 7.19 (t,J=7.2 Hz, 1H), 7.30 (t, J=7.2 Hz, 2H), 7.36 (d, J=7.2 Hz, 2H); ¹³C NMR(CDCl₃, 150 MHz): δ: 24.61, 28.11, 32.97, 34.23, 80.40, 125.97, 128.92,129.25, 136.24, 172.30; HPLC/MS: >95%; HRMS: Calcd. 291.0821, Observed291.0813.

Compound: VNK-I-259; Yield: 71%; Rf: 0.10 DCM-MeOH 100: 1; Mp, ° C.:white powder 67-68; ¹H NMR (CDCl₃, 600 MHz): δ: (DMSO-d₆, 600 MHz), δ1.77 (quintet, J=7.2 Hz, 2H), 2.09 (t, J=7.2 Hz, 2H), 2.95 (t, J=7.2 Hz,2H), 7.10-7.20 (m, 1H), 7.25-7.35 (m, 4H), 11.01 (br.s, 1H); ¹³C NMR(CDCl₃, 150 MHz): δ: 24.90 (CH₂), 31.43 (CH₂), 31.80 (CH₂), 63.61 (CH₃),126.06 (CH), 128.44 (CH), 129.52 (CH), 136.46 (Cq), 168.88 (C═O);HPLC/MS: >95%; HRMS: Calcd. [M+K] 264.0461, Observed 264.0423.

Compound: UPHD-00026; Yield: 78%; Rf; Mp, ° C.: colorless oil; ¹H NMR(CDCl₃, 600 MHz): δ: 1.94 (quintet, J=7.2 Hz, 2H), 2.33 (s, 3H), 2.48(t, J=7.2 Hz, 2H), 2.93 (t, J=7.2 Hz, 2H), 3.68 (s, 3H), 7.12 (d, J=7.8Hz, 2H), 7.28 (d, J=7.8 Hz, 2H); ¹³C NMR (CDCl₃, 150 MHz): δ: 21.02,24.35, 32.59, 33.69, 51.62, 129.72, 130.27, 132.10, 136.30, 173.46;HPLC/MS: >95%; HRMS: n/a.

Compound: UPHD-00022; Yield: 69%; Rf: 0.18 Hex/EA 5:1; Mp, ° C.:colorless oil; ¹H NMR (CDCl₃, 600 MHz): δ: 1.90 (quintet, J=7.2 Hz, 2H),2.46 (t, J=7.2 Hz, 2H), 2.86 (t, J=7.2 Hz, 2H), 3.67 (s, 3H), 3.80 (s,3H), 6.85 (d, J=9.0 Hz, 2H), 7.35 (d, J=9.0 Hz, 2H); ¹³C NMR (CDCl₃, 150MHz): δ: 24.40, 32.54, 35.12, 51.61, 55.33, 114.59, 125.91, 133.40,158.99, 173.50; HPLC/MS: >95%; HRMS: n/a.

Compound: UPHD-00021; Yield: 97%; Rf; Mp, ° C.: colorless oil; ¹H NMR(CDCl₃, 600 MHz): δ: 1.92 (quintet, J=7.2 Hz, 2H), 2.44 (t, J=7.2 Hz,2H), 2.92 (t, J=7.2 Hz, 2H), 3.65 (s, 3H), 7.20-7.26 (m, 4H); ¹³C NMR(CDCl₃, 150 MHz): δ: n/a; HPLC/MS: >95%; HRMS: n/a.

Compound: VNK-I-290; Yield: 98%; Rf: 0.25 Hex/EA 5:1; Mp, ° C.:colorless oil; ¹H NMR (CDCl₃, 600 MHz): δ: 1.94 (quintet, J=7.2 Hz, 2H),2.46 (t, J=7.2 Hz, 2H), 2.94 (t, J=7.2 Hz, 2H), 3.67 (s, 3H), 7.20 (d,J=8.4 Hz, 2H), 7.39 (d, J=8.4 Hz, 2H); ¹³C NMR (CDCl₃, 150 MHz): δ:24.16, 32.51, 32.93, 51.69, 119.85, 130.78, 131.97, 135.28, 173.28;HPLC/MS: >95%; HRMS: Calcd. 288.9898, Observed 288.9873.

Compound: UPHD-00030; Yield: 98%; 0.27; Hex/EA 5:1; Mp, ° C.: colorlessoil; ¹H NMR (CDCl₃, 600 MHz): δ: 1.92 (quintet, J=7.2 Hz, 2H), 2.46 (t,J=7.2 Hz, 2H), 2.91 (t, J=7.2 Hz, 2H), 3.67 (s, 3H), 7.00 (dd, =8.4 Hz,³J_(H-H)=8.4 Hz, 2H), 7.35 (dd, =8.4 Hz, ³J_(H-H)=5.4 Hz, 2H); ¹³C NMR(CDCl₃, 150 MHz): δ: 24.29, 32.49, 34.27, 51.64, 116.05 (d, J_(C-F)=21Hz), 130.73 (d, J_(C-F)=3 Hz), 132.46 (d, J_(C-F)=9 Hz), 161.82 (d,J_(C-F)=224.5 Hz), 173.36; HPLC/MS: >95%; HRMS: n/a.

Compound: UPHD-00028; Yield: 52%; Rf; Mp, ° C.: white solid; 78-79; ¹HNMR (CDCl₃, 600 MHz): δ: 1.94 (quintet, J=7.2 Hz, 2H), 2.34 (s, 3H),2.53 (t, J=7.2 Hz, 2H), 2.95 (t, J=7.2 Hz, 2H), 7.12 (d, J=7.2 Hz, 2H),7.28 (d, J=7.2 Hz, 2H), 10.29 (br.s, 2H); ¹³C NMR (CDCl₃, 150 MHz): δ:21.03, 24.03, 32.21, 33.63, 129.75, 130.41, 131.89, 136.45, 177.61;HPLC/MS: >95%; HRMS: Submitted.

Compound: VNK-I-289; Yield: 71%; Rf: 0.18 Hex/EA 3: 1; Mp, ° C.: whitesolid; 73-75; ¹H NMR (CDCl₃, 600 MHz): δ: 1.90 (quintet, J=7.2 Hz, 2H),2.52 (t, J=7.2 Hz, 2H), 2.88 (t, J=7.2 Hz, 2H), 3.81 (s, 3H), 6.86 (d,J=9.0 Hz, 2H), 7.36 (d, J=9.0 Hz, 2H), 11.36 (br.s, 2H); ¹³C NMR (CDCl₃,150 MHz): δ: 24.06, 32.42, 35.04, 55.34, 114.63, 125.72, 133.52, 159.06,179.12; HPLC/MS: >95%; HRMS: Calcd. 242.0851, Observed 242.1847.

Compound: UPHD-00027; Yield: 51%; Rf; Mp, ° C.: white solid 107-108; ¹HNMR (CDCl₃, 600 MHz): δ: 1.94 (quintet, J=7.2 Hz, 2H), 2.53 (t, J=7.2Hz, 2H), 2.96 (t, J=7.2 Hz, 2H), 7.25-7.27 (m, 4H), 10.95 (br.s, 1H);¹³C NMR (CDCl₃, 150 MHz): δ: 23.85, 32.29, 33.10, 129.11, 130.82,132.21, 134.31, 178.32; HPLC/MS: >95%; HRMS: n/a.

Compound: VNK-I-292; Yield: 56%; Rf; Mp, ° C.: white solid 113-114; ¹HNMR (CDCl₃, 600 MHz): δ: (DMSO-d₆, 600 MHz), δ 1.76 (quintet, J=7.2 Hz,2H), 2.35 (t, J=7.2 Hz, 2H), 2.98 (t, J=7.2 Hz, 2H), 7.28 (d, J=8.4 Hz,2H), 7.48 (d, J=8.4 Hz, 2H), 12.15 (br.s, 2H); ¹³C NMR (CDCl₃, 150 MHz):δ: (DMSO-d₆, 150 MHz), δ 24.39, 31.55, 32.76, 118.83, 130.25, 132.27,136.23, 174.35; HPLC/MS: >95%; HRMS: Calcd. 289.9850 Observed 289.9791.

Compound: UPHD-00029; Yield: 58%; Rf; Mp, ° C.: white solid; ???; ¹H NMR(CDCl₃, 600 MHz): δ: 1.93 (quintet, J=7.2 Hz, 2H), 2.53 (t, J=7.2 Hz,2H), 2.94 (t, J=7.2 Hz, 2H), 7.02 (dd, =8.4 Hz, ³J_(H-H)==8.4 Hz, 2H),7.37 (dd, =8.4 Hz, ³J_(H-H)==5.4 Hz, 2H), 10.74 (br.s, 1H); ¹³C NMR(CDCl₃, 150 MHz): δ: 23.94, 32.49, 34.20, 116.10 (d, J_(C-F)=21 Hz),130.54 (d, J_(C-F)=3 Hz), 132.60 (d, J_(C-F)=7.5 Hz), 161.89 (d,J_(C-F)=246 Hz), 179.53; HPLC/MS: >95%; HRMS: n/a;

Example 8—Testing of Selected PTBA Analogs

Testing of the compounds is on-going, but for those tested, Table 6provides first pass EC₅₀s based upon colormetric in situ hybridizationsfor the Lhx1a mRNA expression levels. After the procedure is run, insitu hybridization is done for the lhx1 mRNA expression and the numberof embryos that show an expanded kidney field is manually counted toestablish the percent of embryos showing expansion. Then the EC₅₀ isdetermined by plotting the percent expansion in Excel, graphing theresults and using the trendline function to determine the 50 percentpoint. The compounds where there is more than one experiment, the EC₅₀is an average of the multiple EC₅₀s generated.

TABLE 6 Renal Cell Expansion and EC₅₀ for selected compounds: Expansionof Cells (including repeats* and recounts**) Compound ID 200 nM 400 nM800 nM 1.5 μM 3 μM EC₅₀ (μM) UPHD-00020 10%  10%  6% 83% 92% 1.4  5% 12% 47%*  84%* 100%* 31% 61% 100%  UPHD-00023 0%  6% 6 92% 100%  1.3  32%89% 100%  UPHD-00029 6% 35% 60% 100%  100%  1.31  6.25%    0% 33% 82.4% (all uncountable) UPHD-00028 3%  9% 61% 97% 100%  11%  16% 56% 100% 100%  UPHD-00027 17% 97% 94% UPHD-00035 25%  10%  0%  0% 21.1%  >3   UPHD-00034 16.7%   10% 35% 53%  0% 0%  9% 14% 29%  9% UPHD-00050 ND ND 0%  0%  0% >3    UPHD-00051 42.1%   25% 35.3%  60% 86.7%  1.9*   6%* 0%*  37%*  45%*  71%* 100%   0% 17% 100%  80% UPHD-00041 ND ND  0%  0% 0% >3    UPHD-00042 10.5%   16.7%   0%  0% 10.5%  >3    52%  69% 69%100%  100%  UPHD-00052 0%  0%  0% 10% 16% >3      0%** UPHD-00067 17% 42% 55% 21% 20% 0.800 19%  15% 15%  0% 30% UPHD-00061 5.5%   0%  0%  6%37% >3 uM UPHD-00059 0%  5% 12% 16% 33% >3 uM UPHD-00069 0%  0%  0%  0% 0% >3 uM UPHD-00070 0%  0%  0%  0%  0% >3 uM UPHD-00024 0% 10% 19% 89%94% 1.27  39% 85% 100%  UPHD-00025 33%  37.5%  21% 89% 97% 0.432 30%* 65%* 81.3%  93.8%  All dead  71%*  95%* All dead* UPHD-00030 22%  26%64% 100%  All dead 0.485 91%  100%  100%  83% All dead 10.5%   36.8% 94.7%  uncountable uncountable UPHD-00021 72% 97% UPHD-00026 3% 14% 49%100%  100%  UPHD-00022 16%   0% 39% 83% 85% 1.4  3% 29% 57% 91% 100% UPHD-00033 ND ND  0%  5% 11% >3 uM UPHD-00032 ND ND  0%  0%  0% >3 uMUPHD-00047 40%*  50%* 100%  100%  100%  0.400 0% 29% 100%* 100%* (14expanded, 28%* 100%* 100%  87% 3 uncountable) 94.4%*  100%* 100%* 100% 100%* UPHD-00048 37%*  69%* 100%  100%  100%  0.280 43%*  50%*  88%* (15expanded, (1 expanded, 67%*  75%* 100%* 4 uncountable) 7 uncountable)100%* 100%* 100%* 100%* UPHD-00090 0% 12% 25% 26% 65% 2.34 UPHD-00040 NDND  6% 15% 45% >3 uM UPHD-00039 ND ND 20% 35% 11% >3 uM UPHD-00045 ND ND17%  0% 17% >3 uM UPHD-00046 ND ND 17% 30% 20% >3 uM (2 expanded, (3expanded, (3 expanded, 10 not expanded, 7 not expanded, 12 not expanded,6 uncountable) 10 uncountable) 5 uncountable) UPHD-00057 19%  19% 11%18% 29% >3 uM UPHD-00063 10%   5%  0%  0%  6% >3 uM UPHD-00062 11%  12% 0%  0%  0% >3 uM UPHD-00058 17%  33% 20% 28%  6% >3 uM UPHD-00065 0%20%  6% 11% 29% >3 uM UPHD-00036 ND ND 45% 85% 100%  UPHD-00038 ND ND58% 81% 85% UPHD-00037 10%*  20%*  0%  0% 10%  2.59**  10%**   0%** 40%*  45%*  80%*  20%**  15%**  60%** UPHD-00049 5% 35% 60% 95% 100% 0.890 UPHD-00053 20%   5% 85% 100%  100%  0.790 UPHD-00077 40%  100% 100%  ND (All Dead) ND (All Dead) 0.230 UPHD-00044 42%* 50% 11% 40% 63% 1.49**  16%**  20%**  55%*  80%* (1 uncountable)  35%**  55%**  95%* 85%** UPHD-00043  0%*  15%*  5% 47% 57% 3*   0%*  5%*  50%* UPHD-000735% 10%  0% 20% 30% >3 uM UPHD-00072 0%  0%  5%  5%  5% >3 uM UPHD-000605%  0% 10% 20%  5% >3 uM UPHD-00066 0%  0%  5% 11% 10% >3 uM UPHD-0007112%  42% 74% 26% 70% UPHD-00081 0% 10% 10% 20% 32% >3 uM

In addition, FIGS. 40A and 40B provide graphs showing EC₅₀s for PTBA(FIG. 40A) and m4PTB (FIG. 40B) done via CNT analysis, essentially asdescribed above on the chd17:EGFP transgene. The results are verysimilar to the EC₅₀s provided in Table 6.

Example 9—HgCl₂-Induced AKI

FIG. 41 is a graph showing the results of an AKI model using HgCl₂induced injury. The assay is a BUN time course of HgCl₂ at 20 mg/kg inBALB/c mice, treated daily after 48 hrs with 3 mg/kg of compoundUPHD-00022 (n=5/group).

Example 10—Folic Acid-Induced AKI

FIG. 42 are photomicrographs showing the results of an AKI model usingfolic acid-induced injury. Folic acid is administered to CD1 mice(n=6/group) intraperitoneally (IP) at 250 mg/ml in 0.3M NaHCO₃. CompoundUPHD-00022 was injected daily IP at 50 mg/ml in 30% PEG300 vehiclestarting 24 hrs after folic acid treatment. Kidney tissue was stainedusing Trichrome and Sirus Red stained after 15 days. Blue in trichromeand the red in sirus red indicates fibrotic (scar) tissue. As can beseen, a reduction in fibrosis is seen.

Example 11—Gentamycin-Induced AKI

FIG. 43A is a graph showing the results of an AKI model usinggentamycin-induced injury. AKI is generated as per a recent publication,Cianciolo Cosenitino, C., et al. ((2010) Intravenous microinjections ofzebrafish larvae to study acute kidney injury. JoVE August 4 (42), pii:2079. doi: 10.3791/2079). Four micromolar m4PTB (UPHD-00025) was addedto the zebrafish E3 media in 0.5% DMSO at 2 days post injection ofgentamicin and the larvae are scored daily for four days (6 days postinjection) as alive or dead, with the final data tallied on day 6 postinjection FIG. 43B.

Example 12—m4PTB Treatment Post AKI: Proliferation

Proliferation of renal cells was examined post AKI for m4PTB inzebrafish larvae (FIG. 44). AKI is generated as above with gentamycin,essentially as described in Example 11. Four μM m4PTB was added 2 dayspost injection of gentamicin. Zebrafish larvae at 5 days post injectionwere treated for 1 hour with the thymidine analogue EdU (10 milimolar),which incorporates into dividing (proliferating cells) while the larvaeare in the zebrafish E3 media. The larvae where then fixed and processedfor histology to count the number of proliferating cells in the proximalconvulted tubule (as marked by the antibody 3G8). The number ofproliferating cells was divided by the total number of cells in theproximal convoluted tubule to generate the percent of proliferation alsoknown as the proliferation index as shown in FIG. 44. Renal cellproliferation is significantly increased in the presence of m4PTB.

Whereas particular embodiments of this invention have been describedabove for purposes of illustration, it will be evident to those skilledin the art that numerous variations of the details of the presentinvention may be made without departing from the invention as defined inthe appended claims.

What is claimed is:
 1. A compound having the formula:

wherein R is S, S(O), S(O)₂ or NH, R1 is —NHR4 where R4 is OH,aminophenyl, hydroxyphenyl, C₁₋₄ alkyl hydroxyphenyl or phenylhydroxyphenyl, or —OR5 where R5 is H or C₁₋₄ alkyl, R2 is phenyl,substituted phenyl,

 where R5 is halo; methyl; C₁₋₄ alkyl; methoxy; C₁₋₄ alkoxy; naphthyl;1H-1,3-benzodiazol-2-yl; 1,3-benzothiazol-2-yl; pyrimidinyl;1-methyl-1H-1,3benzodiazol-2-yl; pyridyl; methoxyphenyl;methylthiophenyl, and R3 is from 0 to 5 methylene groups ((—CH₂-)₀₋₅)and 0 or 1 phenylene wherein at least one methylene or phenylene ispresent, or a pharmaceutically acceptable salt thereof, other than4-(phenylthio)butanoic acid.
 2. The compound of claim 1, wherein R is S.3. The compound of claim 2, wherein R3 is —CH₂—CH₂—CH₂—,—CH₂—CH₂—CH₂—CH₂— or —CH₂—CH₂—CH₂—CH₂—CH₂—.
 4. The compound of claim 2,wherein R2 is phenyl, 4-fluorophenyl, 4-methoxyphenyl or 4-methylphenyl.5. The compound of claim 2, wherein R3 is —CH₂—CH₂—CH₂— or—CH₂—CH₂—CH₂—CH₂—CH₂— and R2 is phenyl, 4-fluorophenyl, 4-methoxyphenylor 4-methylphenyl.
 6. The compound of claim 2, wherein R1 is —NH—OH,—NH-2-aminophenyl, —NH-2-hydroxyphenyl or —O—CH₃.
 7. The compound ofclaim 1, having the formula:

or a pharmaceutically acceptable salt thereof.
 8. The compound of claim1, having the formula:

or a pharmaceutically acceptable salt thereof.
 9. The compound of claim1, having the formula:

or a pharmaceutically acceptable salt thereof.
 10. The compound of claim1, having the formula:

or a pharmaceutically acceptable salt thereof.
 11. The compound of claim1, having the formula:

or a pharmaceutically acceptable salt thereof.
 12. A compositioncomprising a compound having the formula:

wherein R is S, S(O), S(O)₂ or NH, R1 is —NHR4 where R4 is OH,aminophenyl, hydroxyphenyl, C₁₋₄ alkyl hydroxyphenyl or phenylhydroxyphenyl, or —OR5 where R5 is H or C₁₋₄ alkyl, R2 is phenyl,substituted phenyl,

 where R5 is halo; methyl, C₁₋₄ alkyl; methoxy, C₁₋₄ alkoxy; naphthyl;1H-1,3-benzodiazol-2-yl; 1,3-benzothiazol-2-yl; pyrimidinyl;1-methyl-1H-1,3benzodiazol-2-yl; pyridyl; methoxyphenyl;methylthiophenyl, and R3 is from 0 to 5 methylene groups ((—CH₂-)₀₋₅)and 0 or 1 phenylene wherein at least one methylene or phenylene ispresent, or a pharmaceutically acceptable salt thereof, in an amounteffective to increase renal progenitor cell production in a kidney of apatient and a pharmaceutically-acceptable excipient.
 13. The compositionof claim 12, wherein R is S.
 14. The composition of claim 13, wherein R3is —CH₂—CH₂—CH₂—, —CH₂—CH₂—CH₂—CH₂— or —CH₂—CH₂—CH₂—CH₂—CH₂—.
 15. Thecomposition of claim 13, wherein R2 is phenyl, 4-fluorophenyl,4-methoxyphenyl or 4-methylphenyl.
 16. The composition of claim 13,wherein R3 is —CH₂—CH₂—CH₂— or —CH₂—CH₂—CH₂—CH₂—CH₂— and R2 is phenyl,4-fluorophenyl, 4-methoxyphenyl or 4-methylphenyl.
 17. The compositionof claim 13, in which R1 is —NH—OH, —NH-2-aminophenyl,—NH-2-hydroxyphenyl or —O—CH₃.
 18. The composition of claim 12, havingthe formula:

or a pharmaceutically acceptable salt of any of the preceding.
 19. Thecomposition of claim 12, having the formula:

or a pharmaceutically acceptable salt thereof.
 20. The composition ofclaim 12, comprising a cyclodextrin complexed with the compound.